A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

Science.gov (United States)

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant

A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus

NARCIS (Netherlands)

Kool, M.; Goldbach, R. W.; Vlak, J. M.

1994-01-01

In addition to the seven known homologous regions (hrs) of Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindIII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this ori contains several

Enhanced activity of Anticarsia gemmatalis Hüb. (Lepidoptera: Noctuidae) nuclear polyhedrosis virus by boric acid in the laboratory

OpenAIRE

Morales, Lauro; Moscardi, Flávio; Sosa-Gómez, Daniel R.; Paro, Fábio E.; Soldorio, Ivanilda L.

1997-01-01

Boric acid concentrations (0.02,0.03,0.045,0.067 and 0.101 g/100 ml of diet) were evaluated in combination with the Anticarsia gemmatalis Hüb. nuclear polyhedrosis virus (AgNPV) for enhanced virali activity against the insect. Seven days after inoculation, the median lethal concentration (LC50) was 1.52 x 10(5) for the AgNPV alone and 7.95 x 10² for the NPV mixed with 0.045g of boric acid/100 ml of diet. At subsequent evaluation dates (9,11 and 14 days after inoculation) LC50's for NPV+boric ...

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

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Itabashi, Etsuko; Kazama, Tomohiko; Toriyama, Kinya

2009-02-01

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.

Microscopy based studies on the interaction of bio-based silver nanoparticles with Bombyx mori Nuclear Polyhedrosis virus.

Science.gov (United States)

Tamilselvan, Selvaraj; Ashokkumar, Thirunavukkarasu; Govindaraju, Kasivelu

2017-04-01

In the present investigation, silver nanoparticles (AgNPs) interactions with Bombyx mori Nuclear Polyhedrosis virus (BmNPV) were characterized using High-Resolution Scanning Electron Microscopy (HR-SEM), Energy Dispersive X-ray Analysis (EDAX), Transmission Electron Microscopy (TEM), Atomic Force Microcopy (AFM) and Confocal Microscope (CM). HR-SEM study reveals that the biosynthesized AgNPs have interacted with BmNPV and were found on the surface. TEM micrographs of normal and viral polyhedra treated with AgNPs showed that the nanoparticles were accumulated in the membrane and it was noted that some of the AgNPs successfully penetrated the membrane by reaching the capsid of BmNPV. AFM and confocal microscopy studies reveal that the disruption in the shell membrane tends to lose its stability due to exposure of AgNPs to BmNPV. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

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Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

2011-01-01

Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

Mechanisms for cytoplasmic organization: an overview.

Science.gov (United States)

Pagliaro, L

2000-01-01

One of the basic characteristics of life is the intrinsic organization of cytoplasm, yet we know surprisingly little about the manner in which cytoplasmic macromolecules are arranged. It is clear that cytoplasm is not the homogeneous "soup" it was once envisioned to be, but a comprehensive model for cytoplasmic organization is not available in modern cell biology. The premise of this volume is that phase separation in cytoplasm may play a role in organization at the subcellular level. Other mechanisms for non-membrane-bounded intracellular organization have previously been proposed. Some of these will be reviewed in this chapter. Multiple mechanisms, involving phase separation, specific intracellular targeting, formation of macromolecular complexes, and channeling, all could well contribute to cytoplasmic organization. Temporal and spatial organization, as well as composition, are likely to be important in defining the characteristics of cytoplasm.

Nucleic acids synthesis of nuclear polyhedrosis virus in cultured embryonic cells of silkworm

International Nuclear Information System (INIS)

Himeno, Michio; Kimura, Yukio; Hayashiya, Keizo.

1976-01-01

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of 32 P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affected the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C. (auth.)

Cytoplasmic Z-RNA

International Nuclear Information System (INIS)

Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

1987-01-01

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

Cellular Subcompartments through Cytoplasmic Streaming.

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Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

2015-08-24

Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytoplasmic influence of nucleolar development

International Nuclear Information System (INIS)

Ghosh, Sibdas

1974-01-01

The role of cytoplasmic factors on the development of nucleolus in nucleus has been investigated in Ehrlich mouse ascites tumour cells using tritiated thymidine/uridine for autoradiography. It is inferred from the observations that the cytoplasmic factors has some but not absolute control over the development of nucleolus. (M.G.B.)

Cytoplasmic Streaming - Skylab Student Experiment ED-63

Science.gov (United States)

1973-01-01

This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes

Science.gov (United States)

Christie, Joshua R.; Beekman, Madeleine

2017-01-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution. PMID:28025277

Cytoplasmic Estrogen Receptor in breast cancer

Science.gov (United States)

Welsh, Allison W.; Lannin, Donald R.; Young, Gregory S.; Sherman, Mark E.; Figueroa, Jonine D.; Henry, N. Lynn; Ryden, Lisa; Kim, Chungyeul; Love, Richard R.; Schiff, Rachel; Rimm, David L.

2011-01-01

Purpose In addition to genomic signaling, it is accepted that ERα has non-nuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of non-nuclear ER in clinical specimens. Experimental Design A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, 1D5) were validated by western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. Results Four of the antibodies specifically recognized ER by western and QIF, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with pearson’s correlations (r2 values)ranging from 0.87-0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other 4 antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. Conclusions Our data shows ERα present in the cytoplasm in a number of cases using multiple antibodies, while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value. PMID:21980134

Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

International Nuclear Information System (INIS)

Kim, Daeyou; Jeon, Choonju; Kim, Jeong-Hwan; Kim, Mi-Seon; Yoon, Cheol-Hee; Choi, In-Soo; Kim, Sung-Hoon; Bae, Yong-Soo

2006-01-01

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused β-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-β-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

Directory of Open Access Journals (Sweden)

Toshiyuki Takahashi

Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

Science.gov (United States)

Christie, Joshua R; Beekman, Madeleine

2017-03-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis

NARCIS (Netherlands)

Kallenberg, Cees G. M.

Purpose of reviews This review focuses on recent advance in the diagnosis pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Recent findings Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and

The molecular mechanism and physiological role of cytoplasmic streaming.

Science.gov (United States)

Tominaga, Motoki; Ito, Kohji

2015-10-01

Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Science.gov (United States)

Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

2017-10-19

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Cytoplasmic streaming velocity as a plant size determinant.

Science.gov (United States)

Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

2013-11-11

Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants. Copyright © 2013 Elsevier Inc. All rights reserved.

Cytoplasmic Streaming in the Drosophila Oocyte.

Science.gov (United States)

Quinlan, Margot E

2016-10-06

Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

Science.gov (United States)

Haukland, H H; Ulvatne, H; Sandvik, K; Vorland, L H

2001-11-23

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

CTP synthase forms cytoophidia in the cytoplasm and nucleus

International Nuclear Information System (INIS)

Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

2014-01-01

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

CTP synthase forms cytoophidia in the cytoplasm and nucleus

Energy Technology Data Exchange (ETDEWEB)

Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

2014-04-15

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

The Composition and Organization of Cytoplasm in Prebiotic Cells

Directory of Open Access Journals (Sweden)

Jack T. Trevors

2011-03-01

Full Text Available This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1–0.2 µm3 (possibly less if a nanocell prior to the first cell division.

Xenopus egg cytoplasm with intact actin.

Science.gov (United States)

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

Science.gov (United States)

Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

2012-01-01

The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

Directory of Open Access Journals (Sweden)

Antonio Postigo

2017-05-01

Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

Directory of Open Access Journals (Sweden)

Mayumi F. Kohiyama

2015-01-01

Full Text Available Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by a coding polyglutamine expansion in the Ataxin-1 gene ( ATXN1 , which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

First cytoplasmic loop of glucagon-like peptide-1 receptor can function at the third cytoplasmic loop position of rhodopsin.

Science.gov (United States)

Yamashita, Takahiro; Tose, Koji; Shichida, Yoshinori

2008-01-01

G protein-coupled receptors (GPCRs) are classified into several families based on their amino acid sequences. In family 1, GPCRs such as rhodopsin and adrenergic receptor, the structure-function relationship has been extensively investigated to demonstrate that exposure of the third cytoplasmic loop is essential for selective G protein activation. In contrast, much less is known about other families. Here we prepared chimeric mutants between Gt-coupled rhodopsin and Gi/Go- and Gs-coupled glucagon-like peptide-1 (GLP-1) receptor of family 2 and tried to identify the loop region that functions at the third cytoplasmic loop position of rhodopsin. We succeeded in expressing a mutant having the first cytoplasmic loop of GLP-1 receptor and found that this mutant activated Gi and Go efficiently but did not activate Gt. Moreover, the rhodopsin mutant having the first loop of Gs-coupled secretin receptor of family 2 decreased the Gi and Go activation efficiencies. Therefore, the first loop of GLP-1 receptor would share a similar role to the third loop of rhodopsin in G protein activation. This result strongly suggested that different families of GPCRs have maintained molecular architectures of their ancestral types to generate a common mechanism, namely exposure of the cytoplasmic loop, to activate peripheral G protein.

Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

Science.gov (United States)

Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

2017-04-01

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

International Nuclear Information System (INIS)

Symillides, Y.

1985-09-01

An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14 C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments.

Science.gov (United States)

Kumar, Amit; Bhowmick, Prolay Kumar; Singh, Vikram Jeet; Malik, Manoj; Gupta, Ashish Kumar; Seth, R; Nagarajan, M; Krishnan, S Gopala; Singh, Ashok Kumar

2017-10-01

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4 . Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F 1 s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F 1 s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363

Development of novel electrospun nanofibrous scaffold from P. ricini and A. mylitta silk fibroin blend with improved surface and biological properties

International Nuclear Information System (INIS)

Panda, N.; Bissoyi, A.; Pramanik, K.; Biswas, A.

2015-01-01

Biomaterials that stimulate cell attachment and proliferation without any surface modification (e.g. RGD coating) provide potent and cost effective scaffold for regenerative medicine. This study assessed the physico-chemical properties and cell supportive potential of a silk fibroin blend scaffold derived from eri (Philosamia ricini) and tasar (Antheraea mylitta) silk (ET) respectively by electrospinning process. The scanning electron microscopy and transmission electron microscopy study found that the fiber diameters are in 200 to 800 nm range with flat morphology. The porosity of ET scaffold is found to be 79 ± 5% with majority of pore diameter between 2.5 to 5 nm. Similarly, Bombyx mori (BM) silk fibroin and gelatin nanofibrous scaffolds were prepared and taken as control. The ultimate tensile strength of the ET and BM scaffold are found to be 1.83 ± 0.13 MPa and 1.47 ± 0.10 MPa respectively. The measured contact angle (a measure of hydrophilicity) for ET (54.7° ± 1.8°) is found to be lower than BM (62° ± 2.3°). The ability to deposit apatite over ET is comparable to that of BM nanofibers. All the scaffolds were seeded with cord blood derived mesenchymal stem cells (hMSCs) and cultured for 14 days in vitro. The immunofluorescence study reveals enhanced cell attachment with higher metabolic activity for MSCs grown over ET than BM and gelatin. The ET scaffold also demonstrated expression of higher amount cell adhesion molecules (CD29/CD44) and higher proliferation rate than BM and gelatin as confirmed by MTT assay, DNA content estimation assay, flow cytometry study and SEM study. Overall, it may be concluded that ET scaffold may have potential in developing bone tissue grafts for clinical applications in the future. - Highlights: • We have fabricated eri–tasar blended electrospun silk fibroin nanofiber with superior surface property. • The hydrophilicity is higher than the silk fibroin nanofiber derived from Bombyx mori (BM). • The nanofibrous

Development of novel electrospun nanofibrous scaffold from P. ricini and A. mylitta silk fibroin blend with improved surface and biological properties

Energy Technology Data Exchange (ETDEWEB)

Panda, N.; Bissoyi, A.; Pramanik, K.; Biswas, A., E-mail: amitb79@gmail.com

2015-03-01

Biomaterials that stimulate cell attachment and proliferation without any surface modification (e.g. RGD coating) provide potent and cost effective scaffold for regenerative medicine. This study assessed the physico-chemical properties and cell supportive potential of a silk fibroin blend scaffold derived from eri (Philosamia ricini) and tasar (Antheraea mylitta) silk (ET) respectively by electrospinning process. The scanning electron microscopy and transmission electron microscopy study found that the fiber diameters are in 200 to 800 nm range with flat morphology. The porosity of ET scaffold is found to be 79 ± 5% with majority of pore diameter between 2.5 to 5 nm. Similarly, Bombyx mori (BM) silk fibroin and gelatin nanofibrous scaffolds were prepared and taken as control. The ultimate tensile strength of the ET and BM scaffold are found to be 1.83 ± 0.13 MPa and 1.47 ± 0.10 MPa respectively. The measured contact angle (a measure of hydrophilicity) for ET (54.7° ± 1.8°) is found to be lower than BM (62° ± 2.3°). The ability to deposit apatite over ET is comparable to that of BM nanofibers. All the scaffolds were seeded with cord blood derived mesenchymal stem cells (hMSCs) and cultured for 14 days in vitro. The immunofluorescence study reveals enhanced cell attachment with higher metabolic activity for MSCs grown over ET than BM and gelatin. The ET scaffold also demonstrated expression of higher amount cell adhesion molecules (CD29/CD44) and higher proliferation rate than BM and gelatin as confirmed by MTT assay, DNA content estimation assay, flow cytometry study and SEM study. Overall, it may be concluded that ET scaffold may have potential in developing bone tissue grafts for clinical applications in the future. - Highlights: • We have fabricated eri–tasar blended electrospun silk fibroin nanofiber with superior surface property. • The hydrophilicity is higher than the silk fibroin nanofiber derived from Bombyx mori (BM). • The nanofibrous

Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

Energy Technology Data Exchange (ETDEWEB)

Kinoshita, Toshiro [Hokkaido Univ., Sapporo (Japan). Faculty of Agriculture

1982-03-01

Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M/sub 4/ generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets.

Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

International Nuclear Information System (INIS)

Kinoshita, Toshiro

1982-01-01

Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M 4 generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets. (Kaihara, S.)

Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

Science.gov (United States)

Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

Consequences of cytoplasmic irradiation. Studies from microbeam

International Nuclear Information System (INIS)

Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

2009-01-01

The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. (author)

TRIM5α association with cytoplasmic bodies is not required for antiretroviral activity

International Nuclear Information System (INIS)

Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph

2005-01-01

The tripartite motif (TRIM) protein, TRIM5α, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5α. In addition to diffuse cytoplasmic staining, the TRIM5α proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5α from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5α proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5α-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5α fusion proteins indicated that no TRIM5α domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5α

Pediatric Inflammatory Bowel Disease with Cytoplasmic Staining of Antineutrophil Cytoplasmic Antibodies

Directory of Open Access Journals (Sweden)

Omar I. Saadah

2013-01-01

Full Text Available Background. It is unusual for the antineutrophil cytoplasmic antibody with cytoplasmic pattern (cANCA to present in patients with inflammatory bowel disease (IBD without vasculitis. The purpose of this study was to describe the occurrence and characteristics of pediatrics IBD with cANCA. Methods. A retrospective review of pediatric IBD associated with cANCA serology in patients from King Abdulaziz University Hospital, Saudi Arabia, between September 2002 and February 2012. Results. Out of 131 patients with IBD screened for cANCAs, cANCA was positive in 7 (5.3% patients of whom 4 had ulcerative colitis and 3 had Crohn's disease. The median age was 8.8 years (2–14.8 years. Six (86% were males. Of the 7 patients, 5 (71% were Saudi Arabians and 2 were of Indian ethnicity. The most common symptoms were diarrhea, abdominal pain, weight loss, and rectal bleeding. None had family history or clinical features suggestive of vasculitis involving renal and respiratory systems. No difference in the disease location or severity was observed between cANCA positive and cANCA negative patients apart from male preponderance in cANCA positive patients. Conclusion. The occurrence of cANCA in pediatric IBD is rare. Apart from male preponderance, there were no peculiar characteristics for the cANCA positive patients.

Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

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David-Watine Brigitte

2006-05-01

Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

Anticorpos contra o citoplasma de neutrófilos Antineutrophil cytoplasmic antibodies

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Ari Stiel Radu

2005-07-01

Full Text Available A descoberta do marcador sorológico denominado anticorpo anticitoplasma de neutrófilos revolucionou o diagnóstico e o seguimento das vasculites pulmonares, especialmente da granulomatose de Wegener. Seu padrão pode ser citoplasmático e perinuclear. Sua titulação auxilia no diagnóstico e no seguimento das vasculites pulmonares.The discovery of the serological markers known as antineutrophil cytoplasmic antibodies revolutionized the diagnosis and follow-up treatment of the various forms of pulmonary vasculitis, especially that of Wegener's granulomatosis. The antineutrophil cytoplasmic antibodies pattern can be cytoplasmic or perinuclear. Determination of antineutrophil cytoplasmic antibodies titers aids the diagnosis and follow-up treatment of pulmonary vasculitis.

Raman microspectroscopy of nucleus and cytoplasm for human colon cancer diagnosis.

Science.gov (United States)

Liu, Wenjing; Wang, Hongbo; Du, Jingjing; Jing, Chuanyong

2017-11-15

Subcellular Raman analysis is a promising clinic tool for cancer diagnosis, but constrained by the difficulty of deciphering subcellular spectra in actual human tissues. We report a label-free subcellular Raman analysis for use in cancer diagnosis that integrates subcellular signature spectra by subtracting cytoplasm from nucleus spectra (Nuc.-Cyt.) with a partial least squares-discriminant analysis (PLS-DA) model. Raman mapping with the classical least-squares (CLS) model allowed direct visualization of the distribution of the cytoplasm and nucleus. The PLS-DA model was employed to evaluate the diagnostic performance of five types of spectral datasets, including non-selective, nucleus, cytoplasm, ratio of nucleus to cytoplasm (Nuc./Cyt.), and nucleus minus cytoplasm (Nuc.-Cyt.), resulting in diagnostic sensitivity of 88.3%, 84.0%, 98.4%, 84.5%, and 98.9%, respectively. Discriminating between normal and cancerous cells of actual human tissues through subcellular Raman markers is feasible, especially when using the nucleus-cytoplasm difference spectra. The subcellular Raman approach had good stability, and had excellent diagnostic performance for rectal as well as colon tissues. The insights gained from this study shed new light on the general applicability of subcellular Raman analysis in clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Novel Cytoplasmic PARP in the Brain of Octopus vulgaris

Science.gov (United States)

DE LISA, EMILIA; DE MAIO, ANNA; MOROZ, LEONID L.; MOCCIA, FRANCESCO; MENNELLA, MARIA ROSARIA FARAONE; DI COSMO, ANNA

2014-01-01

Recent investigation has focused on the participation of the poly (ADP-ribose) polymerase (PARP) reaction in the invertebrate central nervous system (CNS) during the process of long-term memory (LTM). In this paper, we characterize, localize, and assign a possible role to a cytoplasmic PARP in the brain of Octopus vulgaris. PARP activity was assayed in optic lobes, supraesophageal mass, and optic nerves. The highest levels of enzyme were found in the cytoplasmic fraction. Hyper-activation of the enzyme was detected in Octopus brain after visual discrimination training. Finally, cytoplasmic PARP was found to inhibit Octopus vulgaris actin polymerization. We propose that the cytoplasmic PARP plays a role in vivo to induce the cytoskeletonal reorganization that occurs during learning-induced neuronal plasticity. PMID:22815366

Curious Sex Ratios and Cytoplasmic Genes

Indian Academy of Sciences (India)

instances of curious sex ratios exemplify an important principle: the fitness ..... markable transition - the whole means of sex determination has changed. No longer ... to the cytoplasmic symbiont is self-evident; the symbionts simply increase the.

Magnetite nanoparticles as reporters for microcarrier processing in cytoplasm

Energy Technology Data Exchange (ETDEWEB)

Reibetanz, Uta, E-mail: uta.reibetanz@medizin.uni-leipzig.de [Translational Centre for Regenerative Medicine (TRM) Leipzig, Universitaet Leipzig, Philipp-Rosenthal-Strasse 55, 04103 Leipzig (Germany); Institute for Medical Physics and Biophysics, Medical Faculty, Universitaet Leipzig, Haertelstrasse 16-18, 04107 Leipzig (Germany); Jankuhn, Steffen, E-mail: jankuhn@uni-leipzig.de [Division of Nuclear Solid State Physics, Faculty of Physics and Geosciences, Universitaet Leipzig, Linnestrasse 5, 04103 Leipzig (Germany); Office for Environmental Protection and Occupational Safety, Universitaet Leipzig, Ritterstrasse 24, 04109 Leipzig (Germany)

2011-10-15

The development and therapeutic application of drug delivery systems based on colloidal microcarriers layer-by-layer coated with biopolyelectrolytes requires the investigation of their processing inside the cell for the successful and efficient transport and release of the active agents. The present study is focused on the time-dependent multilayer decomposition and the subsequent release of active agents to the cytoplasm. Magnetite nanoparticles (MNP) were used as reporter agents integrated into the protamine sulfate/dextran sulfate basis multilayer on colloidal SiO{sub 2} cores. This functionalization allows the monitoring of the multilayer decomposition due to the detection of the MNP release, visualized by means of proton-induced X-ray emission (PIXE) by elemental distribution of Si and Fe. The direct correlation between the microcarrier localization in endolysosomes and cytoplasm of HEK293T/17 cells via confocal laser scanning microscopy (CLSM) and the elemental distribution (PIXE) allows tracing the fate of the MNP-coated microcarriers in cytoplasm, and thus the processing of the multilayer. Microcarrier/cell co-incubation experiments of 6 h, 24 h, 48 h, and 72 h show that a MNP release and a slight expansion into the cytoplasm occurs after a longer co-incubation of 72 h.

Detection of antineutrophil cytoplasmic antibodies (ANCAs)

DEFF Research Database (Denmark)

Damoiseaux, Jan; Csernok, Elena; Rasmussen, Niels

2017-01-01

of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF...

Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

International Nuclear Information System (INIS)

Xia, Xi; Weng, Yanjie; Liao, Shujie; Han, Zhiqiang; Liu, Ronghua; Zhu, Tao; Wang, Shixuan; Xu, Gang; Meng, Li; Zhou, Jianfeng; Ma, Ding; Ma, Quanfu; Li, Xiao; Ji, Teng; Chen, Pingbo; Xu, Hongbin; Li, Kezhen; Fang, Yong; Weng, Danhui

2011-01-01

P21 (WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment

Cytoplasmic Control of Sense-Antisense mRNA Pairs

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Flore Sinturel

2015-09-01

Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

Cytoplasmic Control of Sense-Antisense mRNA Pairs.

Science.gov (United States)

Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

2015-09-22

Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Genetic variation among the male sterile cytoplasms induced by gamma irradiation in sugar beets

International Nuclear Information System (INIS)

Mikami, Tetsuo; Kinoshita, Toshiro; Takahashi, Man-emon

1976-01-01

In sugar beets, cytoplasmic male sterility was induced artificially by radiation treatment. In the present study, four kinds of male sterile strain made from the strain H-2002 with normal cytoplasms were used, and the mode of inheritance of the sterility maintained by these strains was confirmed. Also the hereditary mechanism of pollen fructification recovery was studied, and the newly induced heterotypic property of sterile cytoplasms was examined in comparison with naturally found sterile strains. In each of four produced strains, the male sterility was inherited down to M 4 lines stably through mother plants, and it was presumed that the sterility was caused by highly stable cytoplasmic mutation. In each strain, two pairs of nuclear genes took part in the recovery of pollen fructification, but the mode of action of two genes was different. As the result of mating for verification with O type strain to S cytoplasm strain, it seemed that at least the function as O type was not shown to three strains of γ-60, γ-114 and γ-165, and in the sterile cytoplasms of these three strains, the action of fructification recovery genes different from X and Z arose. It was presumed that the genes of X locus did not take effect in these induced cytoplasms. The possibility that at least four kinds of male sterile cytoplasms different from S were induced from normal cytoplasms by artificial mutation was proved indirectly. (Kako, I.)

DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

Curious Sex Ratios and Cytoplasmic Genes

Indian Academy of Sciences (India)

Home; Journals; Resonance – Journal of Science Education; Volume 2; Issue 6. Curious Sex Ratios and Cytoplasmic Genes Microbes Can Distort the Sex Ratio of Populations. Stephen J Freeland Laurence D Hurst. General Article Volume 2 Issue 6 June 1997 pp 68-78 ...

How crowded is the prokaryotic cytoplasm?

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Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

2013-01-01

We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

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Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

2010-01-01

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm.

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

Science.gov (United States)

Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

2015-01-01

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Cytoplasmic Kaiso is associated with poor prognosis in non-small cell lung cancer

International Nuclear Information System (INIS)

Dai, Shun-Dong; Wang, Yan; Miao, Yuan; Zhao, Yue; Zhang, Yong; Jiang, Gui-Yang; Zhang, Peng-Xin; Yang, Zhi-Qiang; Wang, En-Hua

2009-01-01

Kaiso has been identified as a new member of the POZ-zinc finger family of transcription factors that are implicated in development and cancer. Although controversy still exists, Kaiso is supposed to be involved in human cancer. However, there is limited information regarding the clinical significance of cytoplasmic/nuclear Kaiso in human lung cancer. In this study, immunohistochemical studies were performed on 20 cases of normal lung tissues and 294 cases of non-small cell lung cancer (NSCLC), including 50 cases of paired lymph node metastases and 88 cases with complete follow-up records. Three lung cancer cell lines showing primarily nuclear localization of Kaiso were selected to examine whether roles of Kaiso in cytoplasm and in nucleus are identical. Nuclear Kaiso was down-regulated by shRNA technology or addition a specific Kaiso antibody in these cell lines. The proliferative and invasive abilities were evaluated by MTT and Matrigel invasive assay, transcription of Kaiso's target gene matrilysin was detected by RT-PCR. Kaiso was primarily expressed in the cytoplasm of lung cancer tissues. Overall positive cytoplasmic expression rate was 63.61% (187/294). The positive cytoplasmic expression of Kaiso was higher in advanced TNM stages (III+IV) of NSCLC, compared to lower stages (I+II) (p = 0.019). A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found (p = 0.003). In 50 paired cases, cytoplasmic expression of Kaiso was 78.0% (41/50) in primary sites and 90.0% (45/50) in lymph node metastases (p = 0.001). The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%), compared to those with cytoplasmic Kaiso-negative tumors (64.00%) (p = 0.005). Nuclear Kaiso staining was seen in occasional cases with only a 5.10% (15/294) positive rate and was not associated with any clinicopathological features of NSCLC. Furthermore, after the down-regulation of the nuclear

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

International Nuclear Information System (INIS)

Lloyd, Richard E.

2015-01-01

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

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Lloyd, Richard E., E-mail: rlloyd@bcm.edu

2015-05-15

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

Wild Nicotiana Species as a Source of Cytoplasmic Male Sterility in Nicotianatabacum

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Nikova V

2014-12-01

Full Text Available The results of our experiments executed to obtain tobacco male sterile lines through interspecific hybridization are summarized. Ten wild species from the genus Nicotiana: N. excelsior (exc, N. amplexicaulis (amp, N. rustica (rus, Nicotianaglauca (gla, N. velutina (vel, N. benthamiana (ben, N. maritima (mar, N. paniculata (pan, N. longiflora (lon and N. africana (afr were used as cytoplasmic donors and N. tabacum, cv. HarmanliiskaBasma (HB as a donor of the nucleus. Genetic effects of cytoplasmic-nuclear interaction of the studied species are discussed. Our results suggested that cytoplasmic male sterility (CMS was expressed when the cytoplasms of the above mentioned wild Nicotiana species were combined with the nucleus of N. tabacum. The 10 sources of CMS obtained in tobacco were characterized by altered flower phenotypes. Flowers are classified into types according the stamen, pistil and corolla modification. All these CMS sources were backcrossed to Oriental tobaccos, cvs. Tekne, Nevrokop B-12, Kroumovgrad 90 and Djebel 576, to develop corresponding CMS lines. The investigated cytoplasms produced compete male sterility in all those cultivars. The CMS lines preserved flower types, specific for every “sterile” cytoplasm. The extent of male organ modifications varied from apparently normal (but pollenless stamens in CMS (pan, (afr, some plants of (vel (mar through different degrees of malformations (shriveled anther on shortened filaments (lon, pinnate-like anthers on filaments of normal length (amp, petal - (ben, pistil- or stigma-like structures (rus, (gla to lack of male reproductive organs in (exc and in some plants of (vel, (mar, (rus and (gla. Most of the above mentioned cytoplasms had normal female gametophyte and good seed productivity. Alterations of the pistils were observed in CMS (rus, (exc and (ben causing reduction of the seed set. Electrophoresis of seed proteins of the tobacco cultivars and their CMS lines also suggested that

Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

International Nuclear Information System (INIS)

Liu, Yan; Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui; Wen, Jin-kun

2013-01-01

Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs

Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

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Liu, Yan [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); The Third Hospital of Hebei Medical University, Shijazhuang (China); Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); Wen, Jin-kun, E-mail: wjk@hebmu.edu.cn [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China)

2013-06-28

Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.

Baculovirus replication: characterization of DNA and proteins synthesized by a nuclear polyhedrosis virus of Lymantria dispar, the gypsy moth, in a homologous cell line

International Nuclear Information System (INIS)

McClintock, J.T.

1985-01-01

A multiple-embedded nuclear polyhedrosis virus (NPV) of the gypsy moth, Lymantria dispar (LdMNPV), is used for biological control. However, LdMNPV has low natural virulence and a long infection cycle in relation to other NPVs. Therefore, the replicative cycle of LdMNPV was investigated using a homologous cell line, IPLB-LD-652Y. Based on analyses of virus growth curves LdMNPV nonoccluded virus and polyhedral inclusion bodies appeared approximately 20 and 50 hr postinfection (p.i.), respectively. LdMNPV polypeptides, identified by autoradiography of [ 35 S]-methionine labeled fractions in SDS-PAGE, were synthesized in sequential phases: (1) an early α phase of replication (4 polypeptides from 4 to 12 hr p.i.), (2) an intermediate β phase (20 polypeptides from 12 to 24 hr p.i.), and a late γ phase (4 polypeptides from 24 to 28 hr p.i.). In infected cells at least four polypeptides were post-translational cleaved and/or modified. Pulse-labeling with [ 3 H]-mannose, [ 3 H]-N-acetyl-glucosamine or [ 32 P]-monosodium phosphate revealed several viral polypeptides which were glycosylated and/or phosphorylated. DNA:DNA dot hybridization experiments suggested that LdMNPV DNA synthesis was initiated between 12 to 16 hr p.i., increasing significantly thereafter

Cytoplasmic lipid bodies of human neutrophilic leukocytes

International Nuclear Information System (INIS)

Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

1989-01-01

The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

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Tai, Andrew W.; Chuang, Jen-Zen; Sung, Ching-Hwa

2001-01-01

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia. PMID:11425878

Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

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Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

2016-08-01

It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis: two case reports and review of literature

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Spoerl David

2012-12-01

Full Text Available Abstract Background Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis have been described in literature. Case presentation We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient. Conclusion Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis.

Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents.

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Vorob'ev, Vladimir N; Anisimov, Alexander V; Dautova, Nailya R

2008-07-01

The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.

Genetic expression of induced rice sterility under alien-cytoplasm

International Nuclear Information System (INIS)

Wang Naiyuan; Cai Zhijun; Liang Kangjing; Li Yu

2005-01-01

Rice restorer lines were treated with 60 Co γ-ray and 4 male sterile mutants obtained with the fertility of controlled by 4 non-allelic recessive genes, respectively. Sixty combinations were made by using male sterile plants/fertile plants as male parents, and 15 different cytoplasmic substitution lines of the same cell nucleus as female parents. The result showed that F 1 spikelets were normal and fertile, and different numbers of male sterile plants were segregated in F 2 . Complete fertility genotype was not found among interactions between induced male sterile genes and alien-cytoplasms. (authors)

Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

DEFF Research Database (Denmark)

Owens, Trevor; Poole, Ronald J

1979-01-01

Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the...

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

Science.gov (United States)

Hagendoorn, MJM.; Wagner, A. M.; Segers, G.; Van Der Plas, LHW.; Oostdam, A.; Van Walraven, H. S.

1994-10-01

In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

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Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

1987-04-01

Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

Genetic analysis of the cytoplasmic dynein subunit families.

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Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic analysis of the cytoplasmic dynein subunit families.

Directory of Open Access Journals (Sweden)

K Kevin Pfister

2006-01-01

Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

International Nuclear Information System (INIS)

Soldatenkov, V.A.; Filippovich, I.V.

1986-01-01

A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

High viscosity and anisotropy characterize the cytoplasm of fungal dormant stress resistant spores

NARCIS (Netherlands)

Dijksterhuis, J.; Nijsse, J.; Hoekstra, F.A.; Golovina, E.A.

2007-01-01

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia¿the main airborne vehicles of distribution¿are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

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Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2014-01-01

Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

Cytoplasmic genetic variation and extensive cytonuclear interactions influence natural variation in the metabolome

DEFF Research Database (Denmark)

Joseph, Bindu; Corwin, Jason A.; Li, Baohua

2013-01-01

Understanding genome to phenotype linkages has been greatly enabled by genomic sequencing. However, most genome analysis is typically confined to the nuclear genome. We conducted a metabolomic QTL analysis on a reciprocal RIL population structured to examine how variation in the organelle genomes...... was a central hub in the epistatic network controlling the plant metabolome. This epistatic influence manifested such that the cytoplasmic background could alter or hide pairwise epistasis between nuclear loci. Thus, cytoplasmic genetic variation plays a central role in controlling natural variation...... in metabolomic networks. This suggests that cytoplasmic genomes must be included in any future analysis of natural variation....

Evaluation of cytoplasmic genetic effects for production and ...

African Journals Online (AJOL)

uvp

2014-12-03

Dec 3, 2014 ... Cytoplasmic genetic effects are transmitted directly only from mother to offspring through mitochondrial DNA. Normal genetic .... inheritance in three synthetic lines of beef cattle differing in mature size. J. Anim. Sci. 69, 745.

Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

NARCIS (Netherlands)

Rutgers, Abraham; Kallenberg, Cornelis

Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received.

Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

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Funasaka, Tatsuyoshi, E-mail: funasaka@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Balan, Vitaly; Raz, Avraham [Department of Oncology and Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI (United States); Wong, Richard W., E-mail: rwong@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Bio-AFM Frontier Research Center, Kanazawa Kanazawa University, Ishikawa (Japan)

2013-04-26

Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

Amorphous areas in the cytoplasm of Dendrobium tepal cells

Science.gov (United States)

van Doorn, Wouter G.; Kirasak, Kanjana; Ketsa, Saichol

2013-01-01

In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy. PMID:23823702

Cytoplasmic Skp2 expression is associated with p-Akt1 and predicts poor prognosis in human breast carcinomas.

Directory of Open Access Journals (Sweden)

Jing Liu

Full Text Available BACKGROUND: S-phase kinase protein 2 (Skp2, an oncogenic protein, is a key regulator in different cellular and molecular processes, through ubiquitin-proteasome degradation pathway. Increased levels of Skp2 are observed in various types of cancer and associated with poor prognosis. However, in human breast carcinomas, the underlying mechanism and prognostic significance of cytoplasmic Skp2 is still undefined. METHODS: To investigate the role of cytoplasmic Skp2 expression in human breast carcinomas, we immnohistochemically assessed cytoplasmic Skp2, p-Akt1, and p27 expression in 251 patients with invasive ductal carcinomas of the breast. Association of cytoplasmic Skp2 expression with p-Akt1 and p27 was analyzed as well as correspondence with other clinicopathological parameters. Disease-free survival and overall survival were determined based on the Kaplan-Meier method and Cox regression models. RESULTS: Cytoplasmic of Skp2 was detected in 165 out of 251 (65.7% patients. Cytoplasmic Skp2 expression was associated with larger tumor size, more advanced histological grade, and positive HER2 expression. Increased cytoplasmic Skp2 expression correlated with p-Akt1 expression, with 54.2% (51/94 of low p-Akt1-expressing breast carcinomas, but 72.6% (114/157 of high p-Akt1-expressing breast carcinomas exhibiting cytoplasmic Skp2 expression. Elevated cytoplasmic Skp2 expression with low p-Akt1 expression was associated with poor disease-free and overall survival (DFS and OS, and Cox regression models demonstrated that cytoplasmic Skp2 expression was an independent prognostic marker for invasive breast carcinomas. CONCLUSION: Cytoplasmic Skp2 expression is associated with aggressive prognostic factors, such as larger tumor size, and advanced histological grade of the breast cancers. Results demonstrate that combined cytoplasmic Skp2 and p-Akt1 expression may be prognostic for patients with invasive breast carcinomas, and cytoplasmic Skp2 may serve as a

Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

Institute of Scientific and Technical Information of China (English)

Amy L Samuels; Alison Louw; Reza Zareie; Evan Ingley

2017-01-01

AIM To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54.METHODS HEK293 T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate(PMA). Cells were transfected with eG FP-tagged Ankrd54 with or without Lyn tyrosine kinase(wild-type, Y397 F mutant, or Y508 F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagT M gel retardation. Phosphorylated peptides were analysed by multiplereaction-monitoring(MRM) proteomic analysis.RESULTS Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tag TM gel retardation. Co-expression of an active form of the PKCδisoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54(Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased coimmunoprecipitation and enhanced co-localization in the cytoplasm.CONCLUSION We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

Science.gov (United States)

Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

2016-08-01

There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth

Directory of Open Access Journals (Sweden)

Pei-Ying Wang

2018-02-01

Full Text Available The Muscleblind-like protein family (MBNL plays an important role in regulating the transition between differentiation and pluripotency and in the pathogenesis of myotonic dystrophy type 1 (DM1, a CTG expansion disorder. How different MBNL isoforms contribute to the differentiation and are affected in DM1 has not been investigated. Here, we show that the MBNL1 cytoplasmic, but not nuclear, isoform promotes neurite morphogenesis and reverses the morphological defects caused by expanded CUG RNA. Cytoplasmic MBNL1 is polyubiquitinated by lysine 63 (K63. Reduced cytoplasmic MBNL1 in the DM1 mouse brain is consistent with the reduced extent of K63 ubiquitination. Expanded CUG RNA induced the deubiqutination of cytoplasmic MBNL1, which resulted in nuclear translocation and morphological impairment that could be ameliorated by inhibiting K63-linked polyubiquitin chain degradation. Our results suggest that K63-linked ubiquitination of MBNL1 is required for its cytoplasmic localization and that deubiquitination of cytoplasmic MBNL1 is pathogenic in the DM1 brain.

The transmission of cytoplasmic genes in Aspergillus nidulans

NARCIS (Netherlands)

Coenen, A.

1997-01-01

Introduction

This manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces

Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

Science.gov (United States)

Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

2016-01-01

Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

The effect of ionizing irradiation on motion of cytoplasm in cells of Elodea canadensis

International Nuclear Information System (INIS)

Tordyiya, N.V.; Grodzyins'kij, D.M.; Danil'chenko, O.O.

1999-01-01

The effect of acute irradiation on the velocity of cytoplasm is investigated. It is shown that, for small doses, there is a strong nonlinearity between the velocity of cytoplasm and dose. The nonlinear behavior disappears with increasing a dose

Hydrodynamic flow in the cytoplasm of plant cells.

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; Lammeren, A.A. van; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

Hydrodynamic flow in the cytoplasm of plant cells

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; van Lammeren, A.A.M.; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

Hydrodynamic flow in the cytoplasm of plant cells

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; Lammeren, van A.A.M.; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study ( Houtman et al., 2007 )

The poly(rC)-binding protein αCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation

Science.gov (United States)

Vishnu, Melanie R.; Sumaroka, Marina; Klein, Peter S.; Liebhaber, Stephen A.

2011-01-01

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3′ UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3′ UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings. PMID:21444632

Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase

DEFF Research Database (Denmark)

Blostein, R; Daly, S E; MacAulay, Nanna

1998-01-01

This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between...

Mutant p53 protein localized in the cytoplasm inhibits autophagy.

Science.gov (United States)

Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

2008-10-01

The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

Science.gov (United States)

Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

2015-01-01

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Science.gov (United States)

Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

2016-01-01

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

Silicon scaffolds promoting three-dimensional neuronal web of cytoplasmic processes.

Science.gov (United States)

Papadopoulou, Evie L; Samara, Athina; Barberoglou, Marios; Manousaki, Aleka; Pagakis, Stamatis N; Anastasiadou, Ema; Fotakis, Costas; Stratakis, Emmanuel

2010-06-01

Primary neurons were grown on structured silicon (Si) substrates, in the absence of chemotropic factors or synthetic extracellular matrix. The Si substrates used for the study comprise hierarchical structures in the micro- and nanolength scales. The substrates were structured via femtosecond laser irradiation of the Si wafer, in a reactive SF(6) environment. Electron microscopy revealed that the neurons formed an elaborate web of cytoplasmic processes in the absence of glial elements. The neuronal cytoplasm autografted the depth of the spikes, and the neurite sprouting took place over the spikes surface. Here we demonstrate how microfabrication of a Si surface provides an excellent platform for multifaceted studies of neuronal specimens.

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

Science.gov (United States)

Valkenburg, J A; Woldringh, C L

1984-01-01

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid. Images PMID:6389508

Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

Science.gov (United States)

Sander, Suzanne; Arora, Neha; Smith, Emily A

2012-06-01

Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

Science.gov (United States)

Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

2003-07-01

Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

Cytoplasmic tail of coronavirus spike protein has intracellular

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

Science.gov (United States)

Kuang, A.; Musgrave, M. E.

1996-01-01

Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Mieskes, G; Issinger, O G

1993-01-01

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylat......The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation...... and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All...... kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR...

Animal models of antineutrophil cytoplasm antibody-associated vasculitis.

LENUS (Irish Health Repository)

Salama, Alan D

2012-01-01

To provide an update on the experimental models that have been developed recapitulating clinical antineutrophil cytoplasm antibody (ANCA) associated vasculitis. The application of the models in the study of pathogenesis, and the therapeutic implications of this, are covered in the article by van Timmeren and Heeringa in this issue.

Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Directory of Open Access Journals (Sweden)

Mirko eZaffagnini

2013-11-01

Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively-modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively-modified GAPDH in stress signaling pathways in plants.

Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1

International Nuclear Information System (INIS)

Park, EunJoo; Kim, Tae-Houn

2017-01-01

Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1 was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction. - Highlights: • Nuclear and cytoplasmic functions of PYR1 were studied in the mutant which lacked majority of ABA responses. • Nuclear PYR1 reconstituted partially the ABA responses during seed germination, root growth, and guard cell movement. • Both the nuclear and cytoplasmic functions of PYR1 were required for the full generation of ABA responses.

Expression of Anion Exchanger 1 Sequestrates p16 in the Cytoplasm in Gastric, Colonic Adenocarcinoma

Directory of Open Access Journals (Sweden)

Wei-Wei Shen

2007-10-01

Full Text Available p16INK4A (p16 binds to cyclin-dependent kinase 4/6, negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AEi in the cytoplasm in poorly, moderately differentiated gastric, colonic adenocarcinoma cells, in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16, AEi were not detectable. Forced expression of AEi in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AEi in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death, growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric, colonic cancers, we found that 83.33% of gastric cancers, 56.52% of colonic cancers coexpressed AEi, p16 in the cytoplasm. We conclude that AEi plays a crucial role in the pathogenesis of gastric, colonic adenocarcinoma, that p16 dysfunction is a novel pathway of carcinogenesis.

Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS in Nitella Internodal Cells.

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Kenji Kikuchi

Full Text Available Cytoplasmic streaming (CPS is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion.

Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma

Directory of Open Access Journals (Sweden)

Sharmistha Chakraborty

2013-08-01

Full Text Available Tumor necrosis factor receptor 1 (TNFR1-associated death domain protein (TRADD is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM, the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs. PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.

Cytological study of radiation induced alterations in cytoplasmic factors controlling male sterility in corn. Progress report, February 28, 1975--December 1, 1975

International Nuclear Information System (INIS)

Edwardson, J.R.

1975-01-01

Progress is reported on the following research projects: cytoplasmic constituents of the embryo of various gymnosperms and angiosperms; cytoplasmic male sterility in corn; modification of cytoplasmic sterility factors using gamma radiation, EMS, and ethidium bromide; selection for sterile, blight-resistant corn plants; electron microscopy study of abnormal mitochondria in cytoplasm of corn; cytoplasmic male sterility in Petunia; non-Mendelian variegation in Petunia and Nicotiana; graft transmission of cytoplasmic male sterility; cytoplasmic male sterility in Vicia faba; and studies on Blakeslee's I virus in Datura

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

Science.gov (United States)

Xia, J. H.; Roberts, JKM.

1996-05-01

We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

Cytoplasmic male sterility (CMS) in hybrid breeding in field crops.

Science.gov (United States)

Bohra, Abhishek; Jha, Uday C; Adhimoolam, Premkumar; Bisht, Deepak; Singh, Narendra P

2016-05-01

A comprehensive understanding of CMS/Rf system enabled by modern omics tools and technologies considerably improves our ability to harness hybrid technology for enhancing the productivity of field crops. Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. In the context, cytoplasmic male sterility (CMS) owing to its heritable nature to manifest non-functional male gametophyte remains a cost-effective system to promote efficient hybrid seed production. The phenomenon of CMS stems from a complex interplay between maternally-inherited (mitochondrion) and bi-parental (nucleus) genomic elements. In recent years, attempts aimed to comprehend the sterility-inducing factors (orfs) and corresponding fertility determinants (Rf) in plants have greatly increased our access to candidate genomic segments and the cloned genes. To this end, novel insights obtained by applying state-of-the-art omics platforms have substantially enriched our understanding of cytoplasmic-nuclear communication. Concomitantly, molecular tools including DNA markers have been implicated in crop hybrid breeding in order to greatly expedite the progress. Here, we review the status of diverse sterility-inducing cytoplasms and associated Rf factors reported across different field crops along with exploring opportunities for integrating modern omics tools with CMS-based hybrid breeding.

Experimental Analysis of Cell Function Using Cytoplasmic Streaming

Science.gov (United States)

Janssens, Peter; Waldhuber, Megan

2012-01-01

This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

Vasculitis and antineutrophil cytoplasmic autoantibodies associated with propylthiouracil therapy

NARCIS (Netherlands)

Dolman, K. M.; Gans, R. O.; Vervaat, T. J.; Zevenbergen, G.; Maingay, D.; Nikkels, R. E.; Donker, A. J.; von dem Borne, A. E.; Goldschmeding, R.

1993-01-01

Vasculitis is a rare complication of propylthiouracil therapy. Antineutrophil cytoplasmic antibodies (ANCA) have been described in association with several vasculitic disorders. We report detection of ANCA against human neutrophil elastase, proteinase 3, and myeloperoxidase in serum from six

Promising SINEs for embargoing nuclear-cytoplasmic export as an anticancer strategy.

Science.gov (United States)

Tan, David S P; Bedard, Philippe L; Kuruvilla, John; Siu, Lillian L; Razak, Albiruni R Abdul

2014-05-01

In cancer cells, the nuclear-cytoplasmic transport machinery is frequently disrupted, resulting in mislocalization and loss of function for many key regulatory proteins. In this review, the mechanisms by which tumor cells co-opt the nuclear transport machinery to facilitate carcinogenesis, cell survival, drug resistance, and tumor progression will be elucidated, with a particular focus on the role of the nuclear-cytoplasmic export protein. The recent development of a new generation of selective inhibitors of nuclear export (XPO1 antagonists) and how these novel anticancer drugs may bring us closer to the implementation of this therapeutic strategy in the clinic will be discussed.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

Science.gov (United States)

El Hiani, Yassine; Linsdell, Paul

2015-06-19

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of cytoplasmic male sterility (CMS) in perennial ryegrass ( Lolium perenne L.)

DEFF Research Database (Denmark)

Islam, Md. Shofiqul; Møller, Ian Max; Studer, Bruno

2011-01-01

to increase biomass yield, improve nutritional value and tolerance towards abiotic and biotic stress. Cytoplasmic male sterility (CMS) is an efficient tool to control pollination for hybrid seed production. In order to identify the causative polymorphism of the CMS phenotype, a cytoplasmic male sterile plant...

Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

Directory of Open Access Journals (Sweden)

Tomasz J. Wodzicki

2015-01-01

Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence.

Science.gov (United States)

Choi, Jeongjoon; Groisman, Eduardo A

2016-09-01

pH regulates gene expression, biochemical activities and cellular behaviors. A mildly acidic pH activates the master virulence regulatory system PhoP/PhoQ in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The sensor PhoQ harbors an extracytoplasmic domain implicated in signal sensing, and a cytoplasmic domain controlling activation of the regulator PhoP. We now report that, surprisingly, a decrease in Salmonella's own cytoplasmic pH induces transcription of PhoP-activated genes even when the extracytoplasmic pH remains neutral. Amino acid substitutions in PhoQ's cytoplasmic domain hindered activation by acidic pH and attenuated virulence in mice, but did not abolish activation by low Mg(2+) or the antimicrobial peptide C18G. Conversely, removal of PhoQ's extracytoplasmic domains prevented the response to the latter PhoQ-activating signals but not to acidic pH. PhoP-dependent genes were minimally induced by acidic pH in the non-pathogenic species Salmonella bongori but were activated by low Mg(2+) and C18G as in pathogenic S. enterica. Our findings indicate that the sensor PhoQ enables S. enterica to respond to both host- and bacterial-derived signals that alter its cytoplasmic pH. © 2016 John Wiley & Sons Ltd.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

International Nuclear Information System (INIS)

Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

2008-01-01

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

Science.gov (United States)

Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

2010-08-01

Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and

Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

Directory of Open Access Journals (Sweden)

Carolina Wählby

2002-01-01

Full Text Available Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

Science.gov (United States)

Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

2017-01-01

Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

Science.gov (United States)

von Kohn, Christopher; Kiełkowska, Agnieszka; Havey, Michael J

2013-12-01

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

OpenAIRE

Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E

1996-01-01

Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...

Analysis of cytoplasmic effects and fine-mapping of a genic male sterile line in rice.

Directory of Open Access Journals (Sweden)

Peng Qin

Full Text Available Cytoplasm has substantial genetic effects on progeny and is important for yield improvement in rice breeding. Studies on the cytoplasmic effects of cytoplasmic male sterility (CMS show that most types of CMS have negative effects on yield-related traits and that these negative effects vary among CMS. Some types of genic male sterility (GMS, including photo-thermo sensitive male sterility (PTMS, have been widely used in rice breeding, but the cytoplasmic effects of GMS remain unknown. Here, we identified a GMS mutant line, h2s, which exhibited small, white anthers and failed to produce mature pollen. Unlike CMS, the h2s had significant positive cytoplasmic effects on the seed set rate, weight per panicle, yield, and general combining ability (GCA for plant height, seed set rate, weight per panicle, and yield. These effects indicated that h2s cytoplasm may show promise for the improvement of rice yield. Genetic analysis suggested that the phenotype of h2s was controlled by a single recessive locus. We mapped h2s to a 152 kb region on chromosome 6, where 22 candidate genes were predicted. None of the 22 genes had previously been reported to be responsible for the phenotypes of h2s. Sequencing analysis showed a 12 bp deletion in the sixth exon of Loc_Os06g40550 in h2s in comparison to wild type, suggesting that Loc_Os06g40550 is the best candidate gene. These results lay a strong foundation for cloning of the H2S gene to elucidate the molecular mechanism of male reproduction.

Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer.

Science.gov (United States)

Xiao, Lu; Lan, Xiaoying; Shi, Xianping; Zhao, Kai; Wang, Dongrui; Wang, Xuejun; Li, Faqian; Huang, Hongbiao; Liu, Jinbao

2017-05-18

Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.

Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum.

Science.gov (United States)

Lee, Jung-Woo; Shin, Jung-Gul; Kim, Eun Hee; Kang, Hae Eun; Yim, In Been; Kim, Ji Yeon; Joo, Hong-Gu; Woo, Hee Jong

2004-03-01

The immunomodulatory and antitumor effects of lactic acid bacteria (LABs) were investigated. Cytoplasmic fraction of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum were tested for the antiproliferative activity in vitro to SNUC2A, SNU1, NIH/3T3 and Jurkat cell lines by crystal violet assay. All cytoplasmic fraction suppressed proliferation of tumor cells, though L. casei and B. longum were more effective. From these results, cytoplasmic fraction of L. casei and B. longum with Y400 as a control were administered as dietary supplements to Balb/c mice for 2, and 4 consecutive wks. Administration for 4 wks enhanced the number of total T cells, NK cells and MHC class II+ cells, and CD4-CD8+ T cells in flow cytometry analysis. To determine of antitumor activity of LABs preparation in vivo, F9 teratocarcinoma cells were inoculated on mice at 14th day. Body weight was decreased with increased survival rate in all groups with the cytoplasm of LABs. Our results showed that cytoplasmic fraction of LABs had direct antiproliferative effects on tumor cell lines in vitro, effects on immune cells in vivo, and antitumor effects on tumor-bearing mice with prolonged survival periods.

HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

Energy Technology Data Exchange (ETDEWEB)

Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2015-01-30

Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

International Nuclear Information System (INIS)

Kim, Inae; Hur, Jung; Jeong, Sunjoo

2015-01-01

Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells

Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

Science.gov (United States)

Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

2017-11-01

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

Energy Technology Data Exchange (ETDEWEB)

Mahmoud, Nora F. [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Faculty of Pharmacy, Suez Canal University, Ismailia (Egypt); Jasirwan, Chyntia [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Division of Hepatobiliary, Department of Internal Medicine, Faculty of Medicine, University of Indonesia (Indonesia); Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Nagamata, Satoshi [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe (Japan); Kawabata, Akiko [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Tang, Huamin [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Immunology, Nanjing Medical University, Nanjing (China); Mori, Yasuko, E-mail: ymori@med.kobe-u.ac.jp [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan)

2016-03-15

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

International Nuclear Information System (INIS)

Mahmoud, Nora F.; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

2016-01-01

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing.

Science.gov (United States)

Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

2015-01-01

Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection.

Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

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Peng-Cheng Kong

Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

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Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

2016-05-10

The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

pH-Responsive Micelle-Based Cytoplasmic Delivery System for Induction of Cellular Immunity

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Eiji Yuba

2017-11-01

Full Text Available (1 Background: Cytoplasmic delivery of antigens is crucial for the induction of cellular immunity, which is an important immune response for the treatment of cancer and infectious diseases. To date, fusogenic protein-incorporated liposomes and pH-responsive polymer-modified liposomes have been used to achieve cytoplasmic delivery of antigen via membrane rupture or fusion with endosomes. However, a more versatile cytoplasmic delivery system is desired for practical use. For this study, we developed pH-responsive micelles composed of dilauroyl phosphatidylcholine (DLPC and deoxycholic acid and investigated their cytoplasmic delivery performance and immunity-inducing capability. (2 Methods: Interaction of micelles with fluorescence dye-loaded liposomes, intracellular distribution of micelles, and antigenic proteins were observed. Finally, antigen-specific cellular immune response was evaluated in vivo using ELIspot assay. (3 Results: Micelles induced leakage of contents from liposomes via lipid mixing at low pH. Micelles were taken up by dendritic cells mainly via macropinocytosis and delivered ovalbumin (OVA into the cytosol. After intradermal injection of micelles and OVA, OVA-specific cellular immunity was induced in the spleen. (4 Conclusions: pH-responsive micelles composed of DLPC and deoxycholic acid are promising as enhancers of cytosol delivery of antigens and the induction capability of cellular immunity for the treatment of cancer immunotherapy and infectious diseases.

Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS.

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Zhang, Guigen; Chan, Baca; Samarina, Naira; Abere, Bizunesh; Weidner-Glunde, Magdalena; Buch, Anna; Pich, Andreas; Brinkmann, Melanie M; Schulz, Thomas F

2016-02-23

The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.

Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (H. annuus L.)

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Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male-sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred li...

Cytoplasmic male sterility contributes to hybrid incompatibility between subspecies of Arabidopsis lyrata.

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Aalto, Esa A; Koelewijn, Hans-Peter; Savolainen, Outi

2013-10-03

In crosses between evolutionarily diverged populations, genomic incompatibilities may result in sterile hybrids, indicating evolution of reproductive isolation. In several plant families, crosses within a population can also lead to male sterile progeny because of conflict between the maternally and biparentally inherited genomes. We examined hybrid fertility between subspecies of the perennial outcrossing self-incompatible Lyrate rockcress (Arabidopsis lyrata) in large reciprocal F2 progenies and three generations of backcrosses. In one of the reciprocal F2 progenies, almost one-fourth of the plants were male-sterile. Correspondingly, almost one-half of the plants in one of the four reciprocal backcross progenies expressed male sterility. In an additional four independent F2 and backcross families, three segregated male sterility. The observed asymmetrical hybrid incompatibility is attributable to male sterility factors in one cytoplasm, for which the other population lacks effective fertility restorers. Genotyping of 96 molecular markers and quantitative trait locus mapping revealed that only 60% of the plants having the male sterile cytoplasm and lacking the corresponding restorers were phenotypically male-sterile. Genotyping data showed that there is only one restorer locus, which mapped to a 600-kb interval at the top of chromosome 2 in a region containing a cluster of pentatricopeptide repeat genes. Male fertility showed no trade-off with seed production. We discuss the role of cytoplasm and genomic conflict in incipient speciation and conclude that cytoplasmic male sterility-lowering hybrid fitness is a transient effect with limited potential to form permanent reproductive barriers between diverged populations of hermaphrodite self-incompatible species.

Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

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Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

2015-07-01

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

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Christian Much

2016-06-01

Full Text Available Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

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Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

2016-06-01

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

The application of protein markers in conversion of maize inbred lines to the cytoplasmic male sterility basis

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Stevanovic Milan

2016-01-01

Full Text Available A total of seven maize inbred lines of different origin and maturity group were used in the trial set up according to the split-plot randomized complete block design in five environments. Each inbred was observed in five variants: original inbred (N; cytoplasmic male sterile C-type (CMS-C; restorer for CMS-C (RfC; cytoplasmic male sterile S-type (CMS-S and restorer for CMS-S (RfS. The objective was to compare grain yield of original inbreds and their CMS and Rf variants and to apply Isoelectric focusing (IEF to determine whether the conversion of original inbreds to their CMS and Rf counterparts have been done completely. Protein markers have shown that conversion of almost all inbreds was done good and completely. Only original inbreds ZPL2 and ZPL5 did not concur on banding patterns with their RfC variants. The type of cytoplasm had a very significant impact on grain yield. Namely, CMS-C counterparts significantly out yielded their CMS-S versions, while the inbreds with C and S cytoplasm over yielded inbreds with N cytoplasm, as well as their RfC and RfS versions.

Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

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Montserrat Samsó

2009-04-01

Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

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Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

2017-05-01

Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

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Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-07-05

Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

Characterisation and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower

NARCIS (Netherlands)

Spassova, Mariana; Moneger, Françoise; Leaver, Christopher J.; Petrov, Peter; Atanassov, Atanas; Nijkamp, H. John J.; Hille, Jacques

1994-01-01

A new cytoplasmic male sterile sunflower, CMS3, was characterised in relation to the Petiolaris (PET1) cytoplasmic male-sterile sunflower, CMS89. Southern blot analysis showed that the mitochondrial genome of CMS3 contains unique rearrangements in at least five loci (atp6, atp9, atpA, nad1 + 5 and

Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain

International Nuclear Information System (INIS)

Yoshimoto, Nobuo; Tatematsu, Kenji; Koyanagi, Tomoyoshi; Okajima, Toshihide; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

2005-01-01

RBCC protein interacting with PKC 1 (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex

Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

International Nuclear Information System (INIS)

Sauterer, R.A.; Feeney, R.J.; Zieve, G.W.

1988-01-01

Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs

Glucocorticoid control of rat growth hormone gene expression: Effect on cytoplasmic messenger ribonucleic acid production and degradation

International Nuclear Information System (INIS)

Gertz, B.J.; Gardner, D.G.; Baxter, J.D.

1987-01-01

The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of [3H]uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: (1) pulse-chase studies and (2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production

Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

International Nuclear Information System (INIS)

Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

1978-01-01

The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

The effects of moisture and temperature on the ageing kinetics of pollen : Interpretation based on cytoplasmic mobility

NARCIS (Netherlands)

Buitink, J.; Leprince, O.; Hemminga, M.A.; Hoekstra, F.A.

2000-01-01

This study shows that characterization of the molecular mobility in the cytoplasm of pollen provides a new understanding of the effects of moisture and temperature on ageing rates. Using EPR spectroscopy, we determined the rotational motion of the polar spin probe, 3-carboxy-proxyl, in the cytoplasm

Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

OpenAIRE

Carolina Wählby; Joakim Lindblad; Mikael Vondrus; Ewert Bengtsson; Lennart Björkesten

2002-01-01

Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre?processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical ana...

Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

DEFF Research Database (Denmark)

Glahder, Jacob-Andreas Harald; Norrild, Bodil

2011-01-01

Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE......-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR...

α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

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Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

2018-05-02

α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants

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Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversi...

Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

International Nuclear Information System (INIS)

Poznanski, Jaroslaw; Szczesny, Pawel; Ruszczyńska, Katarzyna; Zielenkiewicz, Piotr; Paczek, Leszek

2013-01-01

Highlights: ► We predicted buffering capacity of yeast proteome from protein abundance data. ► We measured total buffering capacity of yeast cytoplasm. ► We showed that proteins contribute insignificantly to buffering capacity. -- Abstract: Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell’s intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

International Nuclear Information System (INIS)

Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

1989-05-01

The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

International Nuclear Information System (INIS)

Whitt, M.A.; Chong, L.; Rose, J.K.

1989-01-01

The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding

AtTZF gene family localizes to cytoplasmic foci

OpenAIRE

Pomeranz, Marcelo; Lin, Pei-Chi; Finer, John; Jang, Jyan-Chyun

2010-01-01

In eukaryotes, mRNA turnover and translational repression represent important regulatory steps in gene expression. Curiously, when under cellular stresses, factors involved in these processes aggregate into cytoplasmic foci known as Processing bodies (P-bodies) and Stress Granules (SGs). In animals, CCCH Tandem Zinc Finger (TZF) proteins play important roles in mRNA decay within P-bodies. TTP, a P-body localized mammalian TZF, can bind to the 3'UTRs of mRNAs containing AU-rich elements (AREs)...

Cytoplasmic expression of survivin is an independent predictor of poor prognosis in patients with salivary gland cancer.

Science.gov (United States)

Stenner, Markus; Weinell, Antje; Ponert, Tobias; Hardt, Aline; Hahn, Moritz; Preuss, Simon F; Guntinas-Lichius, Orlando; Klussmann, Jens Peter

2010-11-01

The expression of the inhibitor of apoptosis protein survivin has been shown to be a significant prognostic indicator in various human cancers. The aim was to assess its expression and prognostic value in salivary gland adenocarcinoma and muco-epidermoid carcinoma. Survivin expression was analysed in 48 patients with parotid gland cancer (21 muco-epidermoid, 27 adenocarcinomas) by means of immunohistochemistry. The experimental findings were correlated with clinicopathological and survival parameters. A high cytoplasmic expression of survivin was found in 30% of the examined tumours without any significant correlation with the patients' clinicopathological characteristics (P > 0.05). Within all patients, the estimated overall survival rate of muco-epidermoid carcinomas was significantly better than that of adenocarcinomas (P = 0.013). A high cytoplasmic survivin expression significantly indicated a poor 5-year disease-free survival rate compared to patients with a low cytoplasmic survivin expression in the whole group (P = 0.001) and in adenocarcinomas (P = 0.004). In a multivariate analysis, a high cytoplasmic survivin expression was the only independent prognostic indicator for a significantly poorer 5-year disease-free survival rate (P = 0.001). The correlation between cytoplasmic survivin expression and survival in salivary gland malignancies might make this an effective tool in patient follow-up, prognosis and targeted therapy in future. © 2010 Blackwell Publishing Limited.

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

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Zhou Li

2011-03-01

Full Text Available Abstract Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Science.gov (United States)

2011-01-01

Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice (Oryza rufipogon

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Xian-hua SHEN

2013-09-01

Full Text Available This study was conducted to develop and characterize a novel cytoplasmic male sterile (CMS source which was identified from Dongxiang wild rice (Oryza rufipogon by crossing Dongxiang wild rice as female with Zhongzao 35, an indica inbred variety, as male and continuous backcrossing with Zhongzao 35. Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm (CMS-WA. Sequential planting showed that this novel CMS has complete and stable male sterility. Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm (CMS-HL and other indica inbred varieties are the maintainers with complete maintaining ability, suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS. So far, we only discovered a unique fertility restoration source for this novel CMS. Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes. Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.

Effect of external pH on the cytoplasmic and vacuolar pHs in Mung bean root-tip cells

International Nuclear Information System (INIS)

Torimitsu, Keiichi; Yazaki, Yoshiaki; Nagasuka, Kinuyo; Ohta, Eiji; Sakata, Makoto

1984-01-01

The effect of the external pH on the intracellular pH in mung bean (Vigna mungo (L.) Hepper) root-tip cells was investigated with the 31 P nuclear magnetic resonance (NMR) method. The 31 P NMR spectra showed three peaks caused by cytoplasmic G-6-P, cytoplasmic Psub(i) and vacuolar Psub(i). The cytoplasmic and vacuolar pHs could be determined by comparing the Psub(i) chemical shifts with the titration curve. When the external pH was changed over a range from pH 3 to 10, the cytoplasmic pH showed smaller changes than the vacuolar pH, suggesting that the former is regulated more strictly than the latter. The H + -ATPase inhibitor, DCCD, caused the breakdown of the mechanism that regulates the intracellular pH. H + -ATPase appears to have an important part in the regulation of the intracellular pH. (author)

Labelling of human resting lymphocytes by continuous infusion of (/sup 3/H)thymidine. 1. Characterization of cytoplasmic label

Energy Technology Data Exchange (ETDEWEB)

Schick, P; Trepel, F; Maisel, K H; Past, W; Reisert, I; Begemann, H; Pilgrim, C [Ulm Univ. (Germany, F.R.)

1978-01-01

After continuous /sup 3/H-TdR infusion in vivo or incubation with /sup 3/H-TdR in vitro human blood lymphocytes were examined by light-microscopic and electron-microscopic autoradiography. Using relatively long autoradiographic exposure times (50-300 days) not only nuclear but also cytoplasmic labelling was visualized, the cytoplasmic label being present in up to 96% of the cells. The cytoplasmic label was predominantly associated with the mitochondria and was removed from the cells nearly completely by treatment with DNase but not with RNase or cold perchloric acid. It is concluded that this cytoplasmic label mainly represents /sup 3/H-TdR incorporated into mitochondrial DNA which is continuously renewed in an average turnover time of 14 days or less. This value is compatible with a turnover time of 11 days for mitochondrial DNA in mammalian cells reported in the literature.

Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

Energy Technology Data Exchange (ETDEWEB)

Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tokyo.ac.jp; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

2016-10-15

Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.

Organelle Simple Sequence Repeat Markers Help to Distinguish Carpelloid Stamen and Normal Cytoplasmic Male Sterile Sources in Broccoli

Science.gov (United States)

Shu, Jinshuai; Liu, Yumei; Li, Zhansheng; Zhang, Lili; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao

2015-01-01

We previously discovered carpelloid stamens when breeding cytoplasmic male sterile lines in broccoli (Brassica oleracea var. italica). In this study, hybrids and multiple backcrosses were produced from different cytoplasmic male sterile carpelloid stamen sources and maintainer lines. Carpelloid stamens caused dysplasia of the flower structure and led to hooked or coiled siliques with poor seed setting, which were inherited in a maternal fashion. Using four distinct carpelloid stamens and twelve distinct normal stamens from cytoplasmic male sterile sources and one maintainer, we used 21 mitochondrial simple sequence repeat (mtSSR) primers and 32 chloroplast SSR primers to identify a mitochondrial marker, mtSSR2, that can differentiate between the cytoplasm of carpelloid and normal stamens. Thereafter, mtSSR2 was used to identify another 34 broccoli accessions, with an accuracy rate of 100%. Analysis of the polymorphic sequences revealed that the mtSSR2 open reading frame of carpelloid stamen sterile sources had a deletion of 51 bases (encoding 18 amino acids) compared with normal stamen materials. The open reading frame is located in the coding region of orf125 and orf108 of the mitochondrial genomes in Brassica crops and had the highest similarity with Raphanus sativus and Brassica carinata. The current study has not only identified a useful molecular marker to detect the cytoplasm of carpelloid stamens during broccoli breeding, but it also provides evidence that the mitochondrial genome is maternally inherited and provides a basis for studying the effect of the cytoplasm on flower organ development in plants. PMID:26407159

Nuclear and Cytoplasmic Delivery of Lactoferrin in Glioma using Chitosan Nanoparticles: Cellular Location Dependent-Action of Lactoferrin.

Science.gov (United States)

Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

2018-05-23

Lactoferrin (Lf) exerts anti-cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti-cancer effect. Upon treatment with 300 and 800 µg/mL Lf loaded chitosan NPs, nuclear targeted Lf-NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf-NPs at 300 µg/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti-cancer protein. Copyright © 2018. Published by Elsevier B.V.

Analysis of genetic effects of nuclear-cytoplasmic interaction on quantitative traits: genetic model for diploid plants.

Science.gov (United States)

Han, Lide; Yang, Jian; Zhu, Jun

2007-06-01

A genetic model was proposed for simultaneously analyzing genetic effects of nuclear, cytoplasm, and nuclear-cytoplasmic interaction (NCI) as well as their genotype by environment (GE) interaction for quantitative traits of diploid plants. In the model, the NCI effects were further partitioned into additive and dominance nuclear-cytoplasmic interaction components. Mixed linear model approaches were used for statistical analysis. On the basis of diallel cross designs, Monte Carlo simulations showed that the genetic model was robust for estimating variance components under several situations without specific effects. Random genetic effects were predicted by an adjusted unbiased prediction (AUP) method. Data on four quantitative traits (boll number, lint percentage, fiber length, and micronaire) in Upland cotton (Gossypium hirsutum L.) were analyzed as a worked example to show the effectiveness of the model.

Molecular analysis of a new cytoplasmic male sterile genotype in sunflower

NARCIS (Netherlands)

Spassova, Mariana; Christov, Michail; Bohorova, Natasha; Petrov, Peter; Dudov, Kalin; Atanassov, Atanas; Nijkamp, H. John J.; Hille, Jaques

1992-01-01

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in

ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

DEFF Research Database (Denmark)

Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

2016-01-01

ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... checkpoints, initiating DNA repair and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

International Nuclear Information System (INIS)

Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

2006-01-01

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16 INK4a , a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

International Nuclear Information System (INIS)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-01-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14 CO 2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle

Antineutrophil cytoplasmic antibody–negative pauci-immune glomerulonephritis with massive intestinal bleeding

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Miyeon Kim

2015-09-01

Full Text Available A 61-year-old woman was admitted to hospital because of generalized edema and proteinuria. Her renal function deteriorated rapidly. Serum immunoglobulin and complement levels were within normal ranges. An autoantibody examination showed negative for antinuclear antibody and antineutrophil cytoplasmic antibody. Histologic examination of a renal biopsy specimen revealed that all of the glomeruli had severe crescent formations with no immune deposits. The patient was treated with steroid pulse therapy with cyclophosphamide followed by oral prednisolone. Fifteen days later, she experienced massive recurrent hematochezia. Angiography revealed an active contrast extravasation in a branch of the distal ileal artery. We selectively embolized with a permanent embolic agent. On the 45th hospital day, the patient suddenly lost consciousness. Brain computed tomography showed intracerebral hemorrhage. We report a case of antineutrophil cytoplasmic antibody–negative pauci-immune glomerulonephritis with massive intestinal bleeding and cerebral hemorrhage.

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

Science.gov (United States)

Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

2018-01-15

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

Science.gov (United States)

Mirza, Sameer; Rakha, Emad A; Alshareeda, Alaa; Mohibi, Shakur; Zhao, Xiangshan; Katafiasz, Bryan J; Wang, Jun; Gurumurthy, Channabasavaiah Basavaraju; Bele, Aditya; Ellis, Ian O; Green, Andrew R; Band, Hamid; Band, Vimla

2013-02-01

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients' outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

Directory of Open Access Journals (Sweden)

Ronald Hancock

Full Text Available Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v or 70 kDa dextran (35% w/v to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox

Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete plant innate immune response.

Directory of Open Access Journals (Sweden)

Ana V García

2010-07-01

Full Text Available An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1. In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.

Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

Science.gov (United States)

Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

2017-04-07

Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytoplasmic male sterility in Petunia hybrida : a structural and histochemical analysis

NARCIS (Netherlands)

Bino, R.J.

1986-01-01

This thesis presents an analysis of the structural and histochemical aspects of cytoplasmic male sterility (cms) in Petuniahybrida . In petunia and in other crops, cms is the most commonly used tool for hybrid seed production. Application of the trait

Usefulness of antineutrophil cytoplasmic autoantibodies in diagnosing and managing systemic vasculitis

NARCIS (Netherlands)

Kallenberg, Cees G. M.

Purpose of reviewAntineutrophil cytoplasmic autoantibodies (ANCAs) are considered important diagnostic tests in the work-up of patients suspected of vasculitis. Here we discuss new developments in the methodology of testing, the pitfalls in using these tests as diagnostic tools, and the value of

Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

Science.gov (United States)

Tortosa, Elena; Hoogenraad, Casper C

2018-02-01

In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evaluation of antineutrophil cytoplasmic antibody seroconversion induced by minocycline, sulfasalazine, or penicillamine

NARCIS (Netherlands)

Choi, HK; Slot, MC; Pan, GL; Weissbach, CA; Niles, JL; Merkel, PA

Objective, Case reports have suggested that minocycline, sulfasalazine, and penicillamine are associated with antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis, This study evaluated ANCA seroconversion due to these agents in serum samples prospectively collected in randomized,

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-03-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization.

Science.gov (United States)

Woodhouse, Francis G; Goldstein, Raymond E

2013-08-27

Many cells exhibit large-scale active circulation of their entire fluid contents, a process termed cytoplasmic streaming. This phenomenon is particularly prevalent in plant cells, often presenting strikingly regimented flow patterns. The driving mechanism in such cells is known: myosin-coated organelles entrain cytoplasm as they process along actin filament bundles fixed at the periphery. Still unknown, however, is the developmental process that constructs the well-ordered actin configurations required for coherent cell-scale flow. Previous experimental works on streaming regeneration in cells of Characean algae, whose longitudinal flow is perhaps the most regimented of all, hint at an autonomous process of microfilament self-organization driving the formation of streaming patterns during morphogenesis. Working from first principles, we propose a robust model of streaming emergence that combines motor dynamics with both microscopic and macroscopic hydrodynamics to explain how several independent processes, each ineffectual on its own, can reinforce to ultimately develop the patterns of streaming observed in the Characeae and other streaming species.

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

NARCIS (Netherlands)

Stroeken, Peter J. M.; Alvarez, Belén; van Rheenen, Jacco; Wijnands, Yvonne M.; Geerts, Dirk; Jalink, Kees; Roos, Ed

2006-01-01

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

Science.gov (United States)

Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

2016-01-01

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

Science.gov (United States)

Sasaki, Kengo; Yamashita, Takahiro; Yoshida, Kazuho; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2014-01-01

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light. PMID:24621599

Cytoplasmic CXCR4 expression in breast cancer: induction by nitric oxide and correlation with lymph node metastasis and poor prognosis

International Nuclear Information System (INIS)

Yasuoka, Hironao; Tsujimoto, Masahiko; Yoshidome, Katsuhide; Nakahara, Masaaki; Kodama, Rieko; Sanke, Tokio; Nakamura, Yasushi

2008-01-01

Lymph nodes constitute the first site of metastasis for most malignancies, and the extent of lymph node involvement is a major criterion for evaluating patient prognosis. The CXC chemokine receptor 4 (CXCR4) has been shown to play an important role in lymph node metastasis. Nitric oxide (NO) may also contribute to induction of metastatic ability in human cancers. CXCR4 expression was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels (a biomarker for peroxynitrate formation from NO in vivo) and lymph node status, CXCR4 immunoreactivity, and other established clinico-pathological parameters, as well as prognosis, was analyzed. Nitrite/nitrate levels and CXCR4 expressions were assessed in MDA-MB-231 and SK-BR-3 breast cancer cell lines after induction and/or inhibition of NO synthesis. CXCR4 staining was predominantly cytoplasmic; this was observed in 50%(56/113) of the tumors. Cytoplasmic CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis. Kaplan-Meier survival curves showed that cytoplasmic CXCR4 expression was associated with reduced disease-free and overall survival. In multivariate analysis, cytoplasmic CXCR4 expression emerged as a significant independent predictor for overall and disease-free survival. Cytoplasmic expression of functional CXCR4 in MDA-MB-231 and SK-BR-3 cells was increased by treatment with the NO donor DETA NONOate. This increase was abolished by L-NAME, an inhibitor of NOS. Our data showed a role for NO in stimulating cytoplasmic CXCR4 expression in vitro. Formation of the biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in vivo. In addition, cytoplasmic CXCR4 expression may serve as a significant prognostic factor for long-term survival in breast cancer

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

International Nuclear Information System (INIS)

Takahashi, Noribumi; Takaiwa, Fumio; Fukuei, Keisuke; Sakamaki, Tadashi; Tanifuji, Shigeyuki

1977-01-01

Incorporation studies with 3 H-uridine or 3 H-adenosine showed that germinating pea embryos synthesize all types of poly A(+) RNA, rRNA and 4-5S RNA at the early stage of germination. After the pulse labeling for 30 min, only heterodisperse RNA and 4-5S RNA appeared in the cytoplasm as labeled RNA species. At this time the radioactivity was associated with cytoplasmic structures heavier than 80S and RNP particles of 68-70S, 52-55S, 36-38S and 20-22S which are presumed to be free mRNP particles in plants. When the pulse-labeled embryos were incubated for a further 60 min in an isotope-free medium, the labeled 17S and 25S rRNA emerged in the cytoplasm, together with labeled heterodisperse and 4-5S RNAs. More radioactivity accumulated in the regions of the polysome, 62-65S and 38-42S particles. The results of analysis of RNAs extracted from the whole cytoplasm, polysome or subribosomal fractions indicated that small subunits of newly formed ribosomes appear more rapidly in the cytoplasm than new large subunits, which accumulate for a while as free particles in the cytoplasm than are incorporated into polysomes. The actinomycin treatment which caused preferential inhibition of rRNA synthesis reduced the accumulation of free, newly formed ribosome subunits and partially permitted detection of the presumed mRNP particles in the subribosomal region even after the chase treatment. (auth.)

A comparative phylogenetic analysis of full-length mariner elements

Indian Academy of Sciences (India)

Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth, Antheraea mylitta with other full length MLEs submitted in the ...

Cytoplasmic streaming in Chara rhizoids: studies in a reduced gravitational field during parabolic flights of rockets.

Science.gov (United States)

Buchen, B; Hejnowicz, Z; Braun, M; Sievers, A

1991-01-01

In-vivo videomicroscopy of Chara rhizoids under 10(-4)g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

Science.gov (United States)

Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-11-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

Science.gov (United States)

Mollenhauer, Hilton H.; Zebrun, William

1960-01-01

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

Plasma exchange in antineutrophil cytoplasmic antibody-associated vasculitis--a 25-year perspective

DEFF Research Database (Denmark)

Szpirt, Wladimir M

2015-01-01

Demonstration of a pathogenic role for antineutrophil cytoplasmic antibodies (ANCA) underlies the scientific rationale for plasma exchange (PLEX) in the treatment of ANCA-associated vasculitis (AAV). Most clinical evidence of efficacy concerns the use of PLEX for the recovery of renal function...

Propylthiouracil-Induced Vasculitis With Antineutrophil Cytoplasmic Antibody.

Science.gov (United States)

Criado, Paulo Ricardo; Grizzo Peres Martins, Ana Claudia; Gaviolli, Camila Fatima; Alavi, Afsaneh

2015-06-01

Propylthiouracil (PTU)-associated vasculitis is a potentially life-threatening disease with a recent increase in the reported cases in the medical literature. This increase may suggest that some earlier cases have been unrecognized or assigned to an alternative nosology category. Although the skin can be the only organ affected by PTU-associated vasculitis, there are many reports with multiple-system involvement. Classically, the symptoms appear under a tetrad of fever, sore throat, arthralgia, and skin lesions. Cutaneous lesions in reported cases of PTU vasculitis have most commonly consisted of retiform acral, purpuric plaques, or nodules. We report a case of perinuclear antineutrophil cytoplasmic antibody-associated vasculitis developed during treatment with PTU for Grave's disease. © The Author(s) 2014.

Comparison of Gene Expression Profiles in Human Germinal Vesicle Before and After Cytoplasmic Transfer From Mature Oocytes in Iranian Infertile Couples

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Fatemeh Sadat Hoseini

2016-08-01

Full Text Available Objective: To evaluate the effect of cytoplasm transfer from mature oocytes to germinal vesicle(GVs on promoting the maturation of cytoplasm of GV at the mRNA level.Materials and methods: Sixty six in vitro fertilization (IVF operations between June 2012 and November 2013 were included in this study. Totally 120 GVs were obtained. Normal GVs were categorized into 3 groups (n = 40 randomly: the first comprised oocytes that did not receive the cytoplasm of mature oocytes; the second group comprised oocytes that did not receive the cytoplasm of mature oocytes but were incubated for 24 h; and the third group comprised oocytes that received 10-15% the cytoplasm of mature oocytes and were then incubated for 24 h. Each group was separately analyzed by quantitative polymerase chain reaction (qPCR and the expression levels of selected genes were assessed.Results: The expression levels of genes involved in the cytoplasmic maturity, and energy-producing mitochondria were significantly higher in the pooled oocytes of 2nd control group than those of the 1st control and intervention groups (p < 0.001. The genes involved in the meiosis, spindle check point, DNA repairing and cell cycle checkpoint did not have any expression in the 1st and intervention groups; however, these genes were expressed in the 2nd group, significantly. In the 2nd group, the highest expression level was observed for genes involved in the DNA repairing and cell cycle checkpoint. In the intervention group, none of the genes were expressed except for energy-producing mitochondria gene; even in this case, the expression level of this gene in this group of oocytes was significantly lower than that in other groups (p < 0.001. After 24 h meiosis assumption was significantly higher in the third group than in the second group (95% vs. 68%, p < 0.001.Conclusion: The cytoplasm transfer technique is not effective in cytoplasmic maturity of the recipient GV oocytes. In contrast, 24-hr in

An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

Science.gov (United States)

Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

2016-01-01

A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing.

Science.gov (United States)

Wang, Zhonghua; Zou, Yanjiao; Li, Xiaoyu; Zhang, Qunyu; Chen, Letian; Wu, Hao; Su, Dihua; Chen, Yuanling; Guo, Jingxin; Luo, Da; Long, Yunming; Zhong, Yang; Liu, Yao-Guang

2006-03-01

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.

Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

DEFF Research Database (Denmark)

Vogel, L K; Norén, Ove; Sjöström, H

1995-01-01

In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance...... of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding.

Directory of Open Access Journals (Sweden)

Koh-ichi Utani

Full Text Available Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

Cytoplasmic BRMS1 expression in malignant melanoma is associated with increased disease-free survival

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Slipicevic Ana

2012-02-01

Full Text Available Abstract Background/aims Breast cancer metastasis suppressor 1 (BRMS1 blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis. Methods Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines. Results A significantly higher percentage of nevi (87%, compared to primary melanomas (20% and metastases (48%, expressed BRMS1 in the nucelus (p Waf1/Cip1 (p = 0.009. Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013 and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033. Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016 and decreased relapse-free period (p = 0.043. Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011, a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro. Conclusion Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion. Please see related article: http://www.biomedcentral.com/1741-7015/10/19

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

Science.gov (United States)

Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

2016-03-01

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation. © 2016 Federation of European Biochemical Societies.

[Role of erythrocyte cytoplasmic structures in changes in the affinity of haemoglobin for oxygen].

Science.gov (United States)

Bryzgalova, N Iu; Brazhe, N A; Iusipovich, A U; Maksimov, G V; Rubin, A B

2009-01-01

Changes in the refractive index of the cytoplasm and the affinity of haemoporphyrin of erythrocyte haemoglobin to oxygen (pH, 2,3-diphosphoglycerate) have been investigated using laser interference microscopy and Raman spectroscopy. It has been established that a decrease in pH and an increase in the content of 2,3-diphosphoglycerate are accompanied by changes in both the form of the cell and the refractive index of the cytoplasm and the affinity of haemoporphyrin of hemoglobin to oxygen. It has been shown that as pH is reduced, the capacity of haemoporphyrin for binding oxygen decreases and as the concentration of 2,3-diphosphoglycerate is increased, the ability of haemoporphyrin for oxygen reabsorption increases.

Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

Science.gov (United States)

Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

2012-05-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

International Nuclear Information System (INIS)

Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

2005-01-01

Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

Directory of Open Access Journals (Sweden)

Makoto Takahashi

Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

Science.gov (United States)

Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

2013-01-01

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

NARCIS (Netherlands)

Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

NARCIS (Netherlands)

Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

Science.gov (United States)

Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

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Takuya Kobayashi

Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes

OpenAIRE

Lu, Fang-Min; Hilgemann, Donald W.

2017-01-01

The Na/K pump exports cytoplasmic Na ions while importing K ions, and its activity is thought to be affected by restricted intracellular Na diffusion in cardiac myocytes. Lu and Hilgemann find instead that the pump can enter an inactivated state and that inactivation can be relieved by cytoplasmic Na.

Cytoplasmic pH and the regulation of the dictyostelium cell cycle

NARCIS (Netherlands)

Aerts, R.J.; Durston, A.J.; Moolenaar, W.H.

1985-01-01

Cytoplasmic pH (pHl) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH “null point” method. There is a cycle of pHl that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S

Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

NARCIS (Netherlands)

Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

2010-01-01

In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

OpenAIRE

Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

1982-01-01

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

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Eugénie Ansseau

Full Text Available Hundreds of double homeobox (DUX genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD. In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain and DUX1 (which is limited to the double homeodomain. Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs. Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs

High ASMA+ Fibroblasts and Low Cytoplasmic HMGB1+ Breast Cancer Cells Predict Poor Prognosis.

Science.gov (United States)

Amornsupak, Kamolporn; Jamjuntra, Pranisa; Warnnissorn, Malee; O-Charoenrat, Pornchai; Sa-Nguanraksa, Doonyapat; Thuwajit, Peti; Eccles, Suzanne A; Thuwajit, Chanitra

2017-10-01

The influence of cancer-associated fibroblasts (CAFs) and high mobility group box 1 (HMGB1) has been recognized in several cancers, although their roles in breast cancer are unclear. The present study aimed to determine the levels and prognostic significance of α-smooth muscle actin-positive (ASMA + ) CAFs, plus HMGB1 and receptor for advanced glycation end products (RAGE) in cancer cells. A total of 127 breast samples, including 96 malignant and 31 benign, were examined for ASMA, HMGB1, and RAGE by immunohistochemistry. The χ 2 test and Fisher's exact test were used to test the association of each protein with clinicopathologic parameters. The Kaplan-Meier method or log-rank test and Cox regression were used for survival analysis. ASMA + fibroblast infiltration was significantly increased in the tumor stroma compared with that in benign breast tissue. The levels of cytoplasmic HMGB1 and RAGE were significantly greater in the breast cancer tissue than in the benign breast tissues. High ASMA expression correlated significantly with large tumor size, clinical stage III-IV, and angiolymphatic and perinodal invasion. In contrast, increased cytoplasmic HMGB1 correlated significantly with small tumor size, pT stage, early clinical stage, luminal subtype (but not triple-negative subtype), and estrogen receptor and progesterone receptor expression. The levels of ASMA (hazard ratio, 14.162; P = .010) and tumor cytoplasmic HMGB1 (hazard ratio, 0.221; P = .005) could serve as independent prognostic markers for metastatic relapse in breast cancer patients. The ASMA-high/HMGB1-low profile provided the most reliable prediction of metastatic relapse. We present for the first time, to the best of our knowledge, the potential clinical implications of the combined assessment of ASMA + fibroblasts and cytoplasmic HMGB1 in breast cancer. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm.

Science.gov (United States)

Kyne, Ciara; Crowley, Peter B

2017-09-19

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal -GR n (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR 5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

Immobilization of bioactive fibroblast growth factor-2 into cubic proteinous microcrystals (Bombyx mori cypovirus polyhedra) that are insoluble in a physiological cellular environment.

Science.gov (United States)

Mori, Hajime; Shukunami, Chisa; Furuyama, Akiko; Notsu, Hiroyuki; Nishizaki, Yuriko; Hiraki, Yuji

2007-06-08

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

Science.gov (United States)

Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

2010-01-20

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic antibodies in coeliac disease before and after gluten-free diet.

Science.gov (United States)

Granito, A; Zauli, D; Muratori, P; Muratori, L; Grassi, A; Bortolotti, R; Petrolini, N; Veronesi, L; Gionchetti, P; Bianchi, F B; Volta, U

2005-04-01

Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies are markers of Crohn's disease and ulcerative colitis respectively. To determine the prevalence of anti-S. cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies in a large series of coeliac disease patients before and after gluten free diet, and to correlate anti-S. cerevisiae-positivity with intestinal mucosal damage. One hundred and five consecutive coeliac disease patients and 141 controls (22 ulcerative colitis, 24 Crohn's disease, 30 primary sclerosing cholangitis, 15 postenteritis syndrome, 50 blood donors) were tested for anti-S. cerevisiae by enzyme-linked immunosorbent assay and for perinuclear anti-neutrophil cytoplasmic autoantibodies by indirect immunofluorescence. In coeliac disease anti-S. cerevisiae (immunoglobulin G and/or immunoglobulin A) were slightly less frequent (59%) than in Crohn's disease (75%, P = 0.16) and significantly more frequent than in ulcerative colitis (27%), primary sclerosing cholangitis (30%), postenteritis syndrome (26%) and blood donors (4%) (P = 0.009, P = 0.0002, P = 0.025, P < 0.0001). No correlation was found between anti-S. cerevisiae and degree of mucosal damage. Perinuclear anti-neutrophil cytoplasmic autoantibodies were detected only in one coeliac. After gluten free diet the disappearance of anti-S. cerevisiae-immunoglobulin A (93%) was more frequent than that of immunoglobulin G (17%, P = 0.0001); perinuclear anti-neutrophil cytoplasmic autoantibodies disappeared in the only coeliac positive at diagnosis. More than half of untreated coeliacs are anti-S. cerevisiae-positive irrespective of the severity of mucosal damage. Differently from immunoglobulin A, anti-S. cerevisiae-immunoglobulin G persisted in more than 80% after gluten free diet. The high prevalence of anti-S. cerevisiae in coeliac disease suggests that they may be the effect of a non-specific immune response in course of chronic small bowel disease.

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

Science.gov (United States)

Zückert, Wolfram R.

2014-01-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization.

Science.gov (United States)

Zhang, Feng; Prahst, Claudia; Mathivet, Thomas; Pibouin-Fragner, Laurence; Zhang, Jiasheng; Genet, Gael; Tong, Raymond; Dubrac, Alexandre; Eichmann, Anne

2016-11-24

Vascular permeability and neovascularization are implicated in many diseases including retinopathies and diabetic wound healing. Robo4 is an endothelial-specific transmembrane receptor that stabilizes the vasculature, as shown in Robo4 -/- mice that develop hyperpermeability, but how Robo4 signals remained unclear. Here we show that Robo4 deletion enhances permeability and revascularization in oxygen-induced retinopathy (OIR) and accelerates cutaneous wound healing. To determine Robo4 signalling pathways, we generated transgenic mice expressing a truncated Robo4 lacking the cytoplasmic domain (Robo4ΔCD). Robo4ΔCD expression is sufficient to prevent permeability, and inhibits OIR revascularization and wound healing in Robo4 -/- mice. Mechanistically, Robo4 does not affect Slit2 signalling, but Robo4 and Robo4ΔCD counteract Vegfr2-Y949 (Y951 in human VEGFR2) phosphorylation by signalling through the endothelial UNC5B receptor. We conclude that Robo4 inhibits angiogenesis and vessel permeability independently of its cytoplasmic domain, while activating VEGFR2-Y951 via ROBO4 inhibition might accelerate tissue revascularization in retinopathy of prematurity and in diabetic patients.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

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Yi-Chieh Lin

Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

Urinary matrix metalloproteinases reflect renal damage in anti-neutrophil cytoplasm autoantibody-associated vasculitis

NARCIS (Netherlands)

Sanders, J.S.F.; Huitema, M.G.; Hanemaaijer, R.; Goor, H. van; Kallenberg, C.G.M.; Stegeman, C.A.

2007-01-01

Renal expression of MMP-2, -9, and tissue inhibitor of MMP-1 (TIMP-1) correlates with histological disease activity in anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV). We studied whether urinary and plasma levels of MMP-2, -9, and TIMP-1 reflect renal expression of these

Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

Energy Technology Data Exchange (ETDEWEB)

Lyi, Sangbom Michael; Tan, Min Jie Alvin, E-mail: tanmja@gis.a-star.edu.sg; Parrish, Colin R., E-mail: crp3@cornell.edu

2014-05-15

Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

International Nuclear Information System (INIS)

Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R.

2014-01-01

Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes

Note: Primer Amysat 001; Fragment size is 211bp

Indian Academy of Sciences (India)

Renuka

Bhandara : Lanes 1–14 represent different strains of Bhandara Ecorace. Note: Primer Amysat 001; Fragment size is 211bp. Fig. 1. SSR profiles generated from genomic DNA of 16 strains from different individuals of (A.L, D. TV, D. BV, Modal, Sukinda, Raily, Bhandara) ecoraces of tasar silk worm, Antheraea mylitta using the.

Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

Science.gov (United States)

Lee, Ok Ran; Cho, Hyung-Taeg

2012-12-01

Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

Cytoplasmic Drosha Is Aberrant in Precancerous Lesions of Gastric Carcinoma and Its Loss Predicts Worse Outcome for Gastric Cancer Patients.

Science.gov (United States)

Zhang, Hailong; Hou, Yixuan; Xu, Liyun; Zeng, Zongyue; Wen, Siyang; Du, Yan-E; Sun, Kexin; Yin, Jiali; Lang, Lei; Tang, Xiaoli; Liu, Manran

2016-04-01

The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume (P = 0.002), tumor grade (P gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.

Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

Science.gov (United States)

Mizejewski, G J

2015-12-01

The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

International Nuclear Information System (INIS)

Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

2008-01-01

The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

Science.gov (United States)

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

2016-05-01

Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Divergent evolution in the cytoplasmic domains of PRLR and GHR genes in Artiodactyla

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Li Meng-Hua

2009-07-01

Full Text Available Abstract Background Prolactin receptor (PRLR and growth hormone receptor (GHR belong to the large superfamily of class 1 cytokine receptors. Both of them have been identified as candidate genes affecting key quantitative traits, like growth and reproduction in livestock. We have previously studied the molecular anatomy of the cytoplasmic domain of GHR in different cattle breeds and artiodactyl species. In this study we have analysed the corresponding cytoplasmic signalling region of PRLR. Results We sequenced PRLR gene exon 10, coding for the major part of the cytoplasmic domain, from cattle, American bison, European bison, yak, sheep, pig and wild boar individuals. We found different patterns of variation in the two receptors within and between ruminants and pigs. Pigs and bison species have no variation within GHR exon 10, but show high haplotype diversity for the PRLR exon 10. In cattle, PRLR shows lower diversity than GHR. The Bovinae PRLR haplotype network fits better the known phylogenetic relationships between the species than that of the GHR, where differences within cattle breeds are larger than between the different species in the subfamily. By comparison with the wild boar haplotypes, a high number of subsequent nonsynonymous substitutions seem to have accumulated in the pig PRLR exon 10 after domestication. Conclusion Both genes affect a multitude of traits that have been targets of selection after domestication. The genes seem to have responded differently to different selection pressures imposed by human artificial selection. The results suggest possible effects of selective sweeps in GHR before domestication in the pig lineage or species divergence in the Bison lineage. The PRLR results may be explained by strong directional selection in pigs or functional switching.

Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

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Johannes, E.; Collings, D. A.; Rink, J. C.; Allen, N. S.; Brown, C. S. (Principal Investigator)

2001-01-01

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

DEFF Research Database (Denmark)

Chen, Jianbo; Grunwald, David; Sardo, Luca

2014-01-01

. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance...... traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag...

Cytoplasmic vitamin A binding proteins in chick embryo dermis and epidermis

International Nuclear Information System (INIS)

Gates, R.E.; King, L.E. Jr.

1985-01-01

Excess vitamin A has striking morphologic and developmental effects on chick embryo skin. While cytoplasmic retinoic acid-binding protein (CRABP) was known to be abundant in chick embryo skin, neither quantitative values nor the distribution between dermis and epidermis have been established. The authors determined CRABP levels in collagenase-separated dermis and epidermis from 8-day-old embryos using specific binding of all-trans-[11- 3 H]retinoic acid in cytosols prepared from gram quantities of these tissues. The level of CRABP in dermis was twice the level in epidermis whether calculated on the basis of wet weight, cytosol protein, or DNA. When averaged over many preparations, 3 times as much dermis as epidermis was recovered from a single piece of skin. Therefore, the dermis contained 85% of the extremely high CRABP levels found in collagenase-treated skin, while epidermis contributed only 15%. Cytoplasmic retinol binding protein (CRBP) was also detected in chick embryo skin, but the binding was low and the levels in epidermis and dermis were not significantly different. The amount of CRABP in chick embryo skin (1600 pmol/g wet weight or 100 pmol/mg cytosol protein) is the highest level reported in any tissue and suggests an important role for vitamin A in the normal development and maturation of skin

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

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Mairaj Ahmed Ansari

2015-07-01

Full Text Available The IL-1β and type I interferon-β (IFN-β molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16 involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1 episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes.

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Lu, Fang-Min; Hilgemann, Donald W

2017-07-03

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when

Binders of intravenously administered zinc 65 in rat liver cytoplasm

International Nuclear Information System (INIS)

Stortenbeek, A.J.; Hamer, C.J.A. van den.

1976-01-01

The fate of intravenously injected trace amounts of 65 Zn 2+ in the rat was studied over a period of ten days after injection. Tissue distributions were determined and a special study was made of 65 Zn-binders in liver cytoplasm. A total of six 65 Zn-binding fractions was found and a tentative identification of the main 65 Zn-binders in these six fractions is given using the collected data regarding their apparent molecular weight, time dependent prominence and content of stable zinc

Cytoplasmic Hu-Antigen R (HuR) Expression is Associated with Poor Survival in Patients with Surgically Resected Cholangiocarcinoma Treated with Adjuvant Gemcitabine-Based Chemotherapy.

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Toyota, Kazuhiro; Murakami, Yoshiaki; Kondo, Naru; Uemura, Kenichiro; Nakagawa, Naoya; Takahashi, Shinya; Sueda, Taijiro

2018-05-01

Hu-antigen R (HuR) is an RNA-binding protein that regulates the stability, translation, and nucleus-to-cytoplasm translocation of messenger RNAs (mRNAs). The aim of this study was to investigate the prognostic significance of HuR in cholangiocarcinoma patients who received adjuvant gemcitabine-based chemotherapy (AGC) after surgical resection. Nuclear and cytoplasmic HuR expression was investigated immunohistochemically in 131 patients with resected cholangiocarcinoma, including 91 patients administered AGC and 40 patients who did not receive adjuvant chemotherapy. The correlation between HuR expression and survival was evaluated by statistical analysis. High nuclear and cytoplasmic HuR expression was observed in 67 (51%) and 45 (34%) patients, respectively. Cytoplasmic HuR expression was significantly associated with lymph node metastasis (p < 0.01), while high cytoplasmic HuR expression was significantly associated with poor disease-free survival [DFS] (p = 0.03) and overall survival [OS] (p = 0.001) in the 91 patients who received AGC, but not in the 40 patients who did not receive AGC (DFS p = 0.17; OS p = 0.07). In the multivariate analysis of patients who received AGC, high cytoplasmic HuR expression was an independent predictor of poor DFS (hazard ratio [HR] 1.77; p = 0.04) and OS (HR 2.09; p = 0.02). Nuclear HuR expression did not affect the survival of enrolled patients. High cytoplasmic HuR expression was closely associated with the efficacy of AGC in patients with cholangiocarcinoma. The current findings warrant further investigations to optimize adjuvant chemotherapy regimens for resectable cholangiocarcinoma.

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

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Yao E Wang

2010-11-01

Full Text Available Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4 pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS and the leucine-rich nuclear export signal (NES found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

International Nuclear Information System (INIS)

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

2013-01-01

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20 NLS mutant gene and examined polysome profile of cells that had been transfected with the S20 NLS gene. As a result, we observed the formation of recombinant 40S carried S20 NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20 NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20 NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20 NLS is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20 NLS . • Cytoplasm-retained S20 NLS is crucial for creating a functional small subunit

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

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Benz, Peter M; Merkel, Carla J; Offner, Kristin; Abeßer, Marco; Ullrich, Melanie; Fischer, Tobias; Bayer, Barbara; Wagner, Helga; Gambaryan, Stepan; Ursitti, Jeanine A; Adham, Ibrahim M; Linke, Wolfgang A; Feller, Stephan M; Fleming, Ingrid; Renné, Thomas; Frantz, Stefan; Unger, Andreas; Schuh, Kai

2013-08-12

In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Cytoplasmic Dynein Promotes HIV-1 Uncoating

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Paulina Pawlica

2014-11-01

Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

A physical perspective on cytoplasmic streaming.

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Goldstein, Raymond E; van de Meent, Jan-Willem

2015-08-06

Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides

NARCIS (Netherlands)

Glasner, Corinna; de Goffau, Marcus C; van Timmeren, Mirjan M; Schulze, Mirja L; Jansen, Benita; Tavakol, Mehri; van Wamel, Willem J B; Stegeman, Coen A; Kallenberg, Cees G M; Arends, Jan P; Rossen, John W; Heeringa, Peter; van Dijl, Jan Maarten

2017-01-01

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides

NARCIS (Netherlands)

C. Glasner (Corinna); M.C. De Goffau (Marcus C.); M.M. Van Timmeren (Mirjan M.); Schulze, M.L. (Mirja L.); Jansen, B. (Benita); M. Tavakol (Mehri); W.J.B. van Wamel (Willem); C.A. Stegeman; C.G.M. Kallenberg (Cees G. M.); J.P.A. Arends (Jan); J.W. Rossen (John); P. Heeringa (Peter); J.M. Dijl (Jan Maarten)

2017-01-01

textabstractThe proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at

Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists[W][OA

Science.gov (United States)

Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen; Meagher, Richard B.

2012-01-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals. PMID:22589468

Cytoplasmic and Genomic Effects on Meiotic Pairing in Brassica Hybrids and Allotetraploids from Pair Crosses of Three Cultivated Diploids

Science.gov (United States)

Cui, Cheng; Ge, Xianhong; Gautam, Mayank; Kang, Lei; Li, Zaiyun

2012-01-01

Interspecific hybridization and allopolyploidization contribute to the origin of many important crops. Synthetic Brassica is a widely used model for the study of genetic recombination and “fixed heterosis” in allopolyploids. To investigate the effects of the cytoplasm and genome combinations on meiotic recombination, we produced digenomic diploid and triploid hybrids and trigenomic triploid hybrids from the reciprocal crosses of three Brassica diploids (B. rapa, AA; B. nigra, BB; B. oleracea, CC). The chromosomes in the resultant hybrids were doubled to obtain three allotetraploids (B. juncea, AA.BB; B. napus, AA.CC; B. carinata, BB.CC). Intra- and intergenomic chromosome pairings in these hybrids were quantified using genomic in situ hybridization and BAC-FISH. The level of intra- and intergenomic pairings varied significantly, depending on the genome combinations and the cytoplasmic background and/or their interaction. The extent of intragenomic pairing was less than that of intergenomic pairing within each genome. The extent of pairing variations within the B genome was less than that within the A and C genomes, each of which had a similar extent of pairing. Synthetic allotetraploids exhibited nondiploidized meiotic behavior, and their chromosomal instabilities were correlated with the relationship of the genomes and cytoplasmic background. Our results highlight the specific roles of the cytoplasm and genome to the chromosomal behaviors of hybrids and allopolyploids. PMID:22505621

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Energy Technology Data Exchange (ETDEWEB)

Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

2011-06-17

Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

International Nuclear Information System (INIS)

Chowdhury, E.H.

2011-01-01

Highlights: → Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. → Fluoridated carbonate apatite promotes a robust increase in transgene expression. → Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

Science.gov (United States)

Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

2014-01-01

ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

Central Diabetes Insipidus in Refractory Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

Science.gov (United States)

Ohashi, Keiji; Morishita, Michiko; Watanabe, Haruki; Sada, Ken-Ei; Katsuyama, Takayuki; Miyawaki, Yoshia; Katsuyama, Eri; Narazaki, Mariko; Tatebe, Noriko; Watanabe, Katsue; Kawabata, Tomoko; Wada, Jun

2017-11-01

We herein describe two cases of refractory antineutrophil cytoplasmic antibody-associated vasculitis (AAV) complicated with diabetes insipidus (DI) possibly related to hypertrophic pachymeningitis (HP). One patient had microscopic polyangiitis and HP, which were refractory to cyclophosphamide, azathioprine, rituximab, mycophenolate mofetil (MMF), and mizoribine. Remission was finally achieved with the use of etanercept, but DI occurred 5 years later. The other patient had granulomatosis with polyangiitis, which that was refractory to cyclophosphamide, methotrexate, MMF, and rituximab. DI subsequently developed, but was successfully treated with etanercept. Dura mater hypertrophy was macroscopically observed in the latter case.

Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

Science.gov (United States)

Beilby, Mary J.

2016-01-01

The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology. PMID:27504112

Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

Directory of Open Access Journals (Sweden)

Hiroyuki eOshiumi

2016-05-01

Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton.

Science.gov (United States)

Suzuki, Hideaki; Rodriguez-Uribe, Laura; Xu, Jiannong; Zhang, Jinfa

2013-10-01

A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9 %) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

Autophagy is involved in the reduction of myelinating Schwann cell cytoplasm during myelin maturation of the peripheral nerve.

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So Young Jang

Full Text Available Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.

Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris

Czech Academy of Sciences Publication Activity Database

Stone, James D.; Koloušková, Pavla; Sloan, D.B.; Štorchová, Helena

2017-01-01

Roč. 68, č. 7 (2017), s. 1599-1612 ISSN 0022-0957 R&D Projects: GA ČR(CZ) GA16-09220S Institutional support: RVO:61389030 Keywords : Cytoplasmic male sterility * Editing * Mitochondrion * Non-coding RNA * Silene vulgaris * Splicing * Transcriptome Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 5.830, year: 2016

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

Science.gov (United States)

Zückert, Wolfram R

2014-08-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.

Science.gov (United States)

Petzoldt, Svenja; Kahra, Dana; Kovermann, Michael; Dingeldein, Artur P G; Niemiec, Moritz S; Ådén, Jörgen; Wittung-Stafshede, Pernilla

2015-06-01

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

Science.gov (United States)

Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2008-07-15

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm.

Science.gov (United States)

Watanabe, J; Kanamura, S

1991-05-01

To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

Energy Technology Data Exchange (ETDEWEB)

Tai, Lin-Ru [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China); Wu, Jing-Ying; Kirby, Ralph [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China)

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction

International Nuclear Information System (INIS)

Corse, Emily; Machamer, Carolyn E.

2003-01-01

Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

International Nuclear Information System (INIS)

Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae; Park, Sang Chul

2010-01-01

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin α, karyopherin β, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

Energy Technology Data Exchange (ETDEWEB)

Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2010-01-01

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

The role of non-coding RNAs in cytoplasmic male sterility in flowering plants

Czech Academy of Sciences Publication Activity Database

Štorchová, Helena

2017-01-01

Roč. 18, č. 11 (2017), č. článku 2429. E-ISSN 1422-0067 R&D Projects: GA ČR GA16-09220S Institutional support: RVO:61389030 Keywords : Cytoplasmic male sterility * Gene expression * Global transcriptome * Non-coding RNA * Pollen development Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 3.226, year: 2016

HuR cytoplasmic expression is associated with increased cyclin A expression and poor outcome with upper urinary tract urothelial carcinoma

International Nuclear Information System (INIS)

Liang, Peir-In; Shiue, Yow-Ling; Huang, Hsuan-Ying; Hsu, Han-Ping; Chen, Li-Tzon; Lin, Ching-Yih; Tai, Chein; Lin, Chun-Mao; Li, Chien-Feng; Li, Wei-Ming; Wang, Yu-Hui; Wu, Ting-Feng; Wu, Wen-Ren; Liao, Alex C; Shen, Kun-Hung; Wei, Yu-Ching; Hsing, Chung-Hsi

2012-01-01

HuR is an RNA-binding protein that post-transcriptionally modulates the expressions of various target genes implicated in carcinogenesis, such as CCNA2 encoding cyclin A. No prior study attempted to evaluate the significance of HuR expression in a large cohort with upper urinary tract urothelial carcinomas (UTUCs). In total, 340 cases of primary localized UTUC without previous or concordant bladder carcinoma were selected. All of these patients received ureterectomy or radical nephroureterectomy with curative intents. Pathological slides were reviewed, and clinical findings were collected. Immunostaining for HuR and cyclin A was performed and evaluated by using H-score. The results of cytoplasmic HuR and nuclear cyclin A expressions were correlated with disease-specific survival (DSS), metastasis-free survival (MeFS), urinary bladder recurrence-free survival (UBRFS), and various clinicopathological factors. HuR cytoplasmic expression was significantly related to the pT status, lymph node metastasis, a higher histological grade, the pattern of invasion, vascular and perineurial invasion, and cyclin A expression (p = 0.005). Importantly, HuR cytoplasmic expression was strongly associated with a worse DSS (p < 0.0001), MeFS (p < 0.0001), and UBRFS (p = 0.0370) in the univariate analysis, and the first two results remained independently predictive of adverse outcomes (p = 0.038, relative risk [RR] = 1.996 for DSS; p = 0.027, RR = 1.880 for MeFS). Cyclin A nuclear expression was associated with a poor DSS (p = 0.0035) and MeFS (p = 0.0015) in the univariate analysis but was not prognosticatory in the multivariate analyses. High-risk patients (pT3 or pT4 with/without nodal metastasis) with high HuR cytoplasmic expression had better DSS if adjuvant chemotherapy was performed (p = 0.015). HuR cytoplasmic expression was correlated with adverse phenotypes and cyclin A overexpression and also independently predictive of worse DSS and MeFS, suggesting its roles in

CONTINUOUS MEASUREMENT OF THE CYTOPLASMIC PH IN LACTOCOCCUS-LACTIS WITH A FLUORESCENT PH INDICATOR

NARCIS (Netherlands)

MOLENAAR, D; ABEE, T; KONINGS, WN

1991-01-01

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the

Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane

NARCIS (Netherlands)

Wenzel, M.; Kohl, B.; Münch, D.; Raatschen, N.; Albada, H.B.; Hamoen, L.; Metzler-Nolte, N.; Sahl, H.G.; Bandow, J.E.

2012-01-01

Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end

Effect of drought stress on male fertility restoration in A3 CMS-inducing cytoplasm of sorghum

Directory of Open Access Journals (Sweden)

Valentin V. Kozhemyakin

2017-08-01

Full Text Available Use of cytoplasmic male sterility (CMS in hybrid breeding requires effective male fertility-restoring lines. In sorghum, very few restoring lines that can restore fertility in A3 CMS have been reported. To identify the reasons for this deficiency, F1 and F2 hybrids of an A3 CMS line crossed with the line IS1112C, a donor of fertility-restoring (Rf genes for A3 cytoplasm, and testcrosses of fertile plants to A3 CMS lines were grown under contrasting water availability regimes in dryland and irrigated field plots. In the irrigated plots the frequency of fertile plants in testcrosses was twice that in dryland plots (P < 0.05. Fertile plants from the F2 family grown in the irrigated plots showed significantly higher restoration ability than fertile plants from the same family grown in dryland plots. F3 plants from the F2 family grown in irrigated plots yielded on average a sixfold higher frequency of fertile plants in testcrosses than F3 plants derived from dryland plots (P < 0.01. Fertility of testcross hybrids correlated negatively with air vapor pressure deficit (VPD at flowering (r = −0.96; P < 0.01 suggesting that VPD is a trigger for downregulation of Rf genes for A3 cytoplasm.

Recent advances in anti-neutrophil cytoplasmic antibody-associated vasculitis

Directory of Open Access Journals (Sweden)

B Lazarus

2016-01-01

Full Text Available Anti-neutrophil cytoplasmic antibody-associated vasculitis is an uncommon inflammatory disease of small to medium-sized vessels that frequently presents with rapidly progressive glomerulonephritis and renal failure though it can affect any organ system. If untreated, the vast majority of patients will die within a year. Current treatments improve prognosis but affected patients remain at a substantially higher risk of death and adverse outcomes. We review the classification of the disease, our understanding of the pathogenesis and epidemiology, and propose future directions for research. We also evaluate the evidence supporting established treatment regimens and the progress of clinical trials for newer treatments to inform the design of future studies.

p25alpha relocalizes in oligodendroglia from myelin to cytoplasmic inclusions in multiple system atrophy

DEFF Research Database (Denmark)

Song, Yun Ju C; Lundvig, Ditte M S; Huang, Yue

2007-01-01

cytoplasmic inclusions. Overall, the data indicate that changes in the cellular interactions between MBP and p25alpha occur early in MSA and contribute to abnormalities in myelin and subsequent alpha-synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease....

Theodor and Marcella Boveri: chromosomes and cytoplasm in heredity and development.

Science.gov (United States)

Satzinger, Helga

2008-03-01

The chromosome theory of heredity, developed in 1902-1904, became one of the foundation stones of twentieth-century genetics. It is usually referred to as the Sutton-Boveri theory after Walter Sutton and Theodor Boveri. However, the contributions of Theodor Boveri and his co-worker, Marcella O'Grady Boveri (also his wife), to the understanding of heredity and development go beyond the localization of the Mendelian hereditary factors onto the chromosomes. They investigated the interaction of cytoplasm and chromosomes, and demonstrated its relevance in heredity and development.

Contribution of the actomyosin motor to the temperature-dependent translational diffusion of water by cytoplasmic streaming in Elodea canadensis cells.

Science.gov (United States)

Vorob'ev, V N; Anisimov, A V; Dautova, N R

2004-12-01

The extent to which the actomyosin motor responsible for cytoplasmic streaming contributes to the translational diffusion of water in Elodea canadensis cells was studied by a nuclear magnetic resonance (NMR) spin-echo technique. The relative contribution of the actomyosin motor was determined from the corresponding apparent diffusion coefficient by the Einstein-Smolukhovsky relation. It is equal to the difference between the diffusional displacements of the cytoplasmic and the bulk water (deltaX). The NMR data show that the temperature dependence of deltaX is humpshaped, which is characteristic of enzyme reactions. At the same time, the apparent diffusion coefficient of cytoplasmic water increases with an increase in temperature. The most significant contribution of the actomyosin motor to deltaX is observed at temperatures below 20 degrees C. Within the temperature range of 20 to 33 degrees C, deltaX changes only slightly, and a further increase in temperature reduces deltaX to zero.

The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes

International Nuclear Information System (INIS)

Greer, Amy E.; Hearing, Patrick; Ketner, Gary

2011-01-01

The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 k in viral late mRNA accumulation.

An Extremely Halophilic Proteobacterium Combines a Highly Acidic Proteome with a Low Cytoplasmic Potassium Content*

Science.gov (United States)

Deole, Ratnakar; Challacombe, Jean; Raiford, Douglas W.; Hoff, Wouter D.

2013-01-01

Halophilic archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant and have evolved highly acidic proteomes that function only at high salinity. We examined osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila and Halorhodospira halochloris. Genome sequencing and isoelectric focusing gel electrophoresis showed that the proteome of H. halophila is acidic. In line with this finding, H. halophila accumulated molar concentrations of KCl when grown in high salt medium as detected by x-ray microanalysis and plasma emission spectrometry. This result extends the taxonomic range of organisms using KCl as a main osmoprotectant to the Proteobacteria. The closely related organism H. halochloris does not exhibit an acidic proteome, matching its inability to accumulate K+. This observation indicates recent evolutionary changes in the osmoprotection strategy of these organisms. Upon growth of H. halophila in low salt medium, its cytoplasmic K+ content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. First, we conclude that proteome acidity is not driven by stabilizing interactions between K+ ions and acidic side chains but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. Second, we propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K+-binding sites on an increasingly acidic protein surface. PMID:23144460

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

International Nuclear Information System (INIS)

Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

2007-01-01

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions

Heritable effect of plant water availability conditions on restoration of male fertility in the ‘9E’ CMS-inducing cytoplasm of sorghum

Directory of Open Access Journals (Sweden)

Lev Aleksandrovich Elkonin

2012-05-01

Full Text Available Heritable changes of phenotype arising in plant ontogenesis by the influence of environmental factors belong to the most intriguing genetic phenomena. Studying restoration of male fertility in the ‘9E’ type of CMS-inducing cytoplasm of sorghum and related CMS-inducing cytoplasms, A4 and M35-1A, in some hybrid combinations, we found an unusual inheritance pattern: the Rf-genes functioned in the self-pollinated progenies of F1 hybrids (up to F10 but did not or poorly expressed in backcrosses of these hybrids to CMS-lines with the same cytoplasm type. In experiments on parallel growing of the same F1 hybrid combinations in the ‘dry plot’ and in the ‘irrigated plot’, it was found that high level of plant water availability during panicle and pollen developmental stages significantly increased male fertility of F1 and testcross hybrid populations, in which fertility-restoring genes were in heterozygote state, whereas in F2 populations the influences of water availability conditions cause less pronounce effects. Similarly, male-sterile F1 plants, being transferred from the ‘dry plot’ to greenhouse, produced male-fertile panicles. In addition, male-sterile plants from F2 families, which segregated-out as recessives, being transferred to greenhouse also produced male-fertile panicles. In the progenies of these revertants that were grown in field conditions and in the ‘dry plot’, stable inheritance of male fertility for 3 cycles of self-pollination was observed, and a number of stable fertile lines in the ‘9E’ cytoplasm were obtained. However, in test-crosses of these fertile lines to CMS-lines with the ‘9E’ cytoplasm restoration of male fertility was not observed, except the progeny of one revertant that behaved as fertility-restorer line. These data suggest that the functional state of fertility-restoring genes for the ‘9E’ sorghum cytoplasm is epigenetically-regulated trait established by the influence of environmental

High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

NARCIS (Netherlands)

Kain, Renate; Tadema, Henko; McKinney, Eoin F.; Benharkou, Alexandra; Brandes, Ricarda; Peschel, Andrea; Hubert, Virginie; Feenstra, Tjerk; Sengoelge, Guerkan; Stegeman, Coen; Heeringa, Peter; Lyons, Paul A.; Smith, Kenneth G. C.; Kallenberg, Cees; Rees, Andrew J.

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti neutrophil cytoplasmic antibody (ANCA) associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We

Uptake and degradation of cytoplasmic RNA by lysosomes in the perfused rat liver

International Nuclear Information System (INIS)

Heydrick, S.J.; Lardeux, B.; Mortimore, G.E.

1987-01-01

The release of [ 14 C]cytidine has been shown previously to be a valid marker for RNA degradation in rat hepatocytes. The breakdown of RNA measured with this marker in perfused livers prelabeled in vivo with [6- 14 C]orotic acid was found to be regulated acutely by perfusate amino acids over a wide range, from 0.29 to 3.48%/h. This regulation paralleled that of lysosomal proteolysis. Chloroquine inhibited RNA degradation 60-70%. In subsequent cell fractionation studies labelled cytidine was released; the distribution of this release paralleled that of a lysosomal marker enzyme. The release plateaued after two hours, defining a distinct lysosomal pool of RNA. The lysosomal location of the RNA pool was confirmed in experiments where a 22% increase in the apparent pool size was obtained by lowering the homogenate pH from 7.0 to 5.5. The pool size correlated linearly with the rate of RNA degradation measured during perfusion, giving a turnover constant in reasonable agreement with values reported for autophagy. These results indicate that cytoplasmic RNA degradation occurs primarily in the lysosome and is regulated under these conditions by the amino acid control of lysosomal sequestration of cytoplasm

Hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis presenting with a vasculitic syndrome, acute nephritis and a puzzling skin rash: a case report

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Keasberry Justin

2013-01-01

Full Text Available Abstract Introduction Anti-neutrophil cytoplasmic antibody-associated vasculitis has been associated with many drugs and it is a relatively rare side effect of the antihypertensive drug hydralazine. The diagnosis and management of patients who have anti-neutrophil cytoplasmic antibody-associated vasculitis may be challenging because of its relative infrequency, variability of clinical expression and changing nomenclature. The spectrum of anti-neutrophil cytoplasmic antibody-associated vasculitis is wide and can be fatal. This case documents a 62-year-old woman who presented with hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis with a puzzling cutaneous rash. Case presentation We report a rare case of hydralazine-induced anti-neutrophil cytoplasmic antibody-associated vasculitis in a 62-year-old Caucasian woman who presented with a vasculitic syndrome with a sore throat, mouth ulcers and otalgia after several months of constitutional symptoms. She then proceeded to develop a rash over her right lower limb. Clinically, the rash had features to suggest Sweet’s syndrome, but also had some appearances consistent with embolic phenomena and did not have the appearance of palpable purpure usually associated with cutaneous vasculitis. Differential diagnoses were hydralazine-associated Sweet’s syndrome, streptococcal-induced cutaneous eruption or an unrelated contact dermatitis. A midstream urine sample detected glomerular blood cells in the setting of anti-neutrophil cytoplasmic antibody-positive renal vasculitis and Streptococcus pyogenes bacteremia. A renal biopsy revealed a pauci-immune, focally necrotizing glomerulonephritis with small crescents. Her skin biopsy revealed a heavy neutrophil infiltrate involving the full thickness of the dermis with no evidence of a leucocytoclastic vasculitis, but was non-specific. She was initially commenced on intravenous lincomycin for her bloodstream infection and subsequently

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

Science.gov (United States)

Fitzgerald, Kerry D; Semler, Bert L

2013-09-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

Science.gov (United States)

Fitzgerald, Kerry D.; Semler, Bert L.

2013-01-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

Potential role for MATER in cytoplasmic lattice formation in murine oocytes.

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Boram Kim

2010-09-01

Full Text Available Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton insoluble structures termed the oocyte cytoplasmic lattices (CPLs. Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function.Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater(tm/tm oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater(+/+ and Mater(tm/tm germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/tm oocytes compared to Mater(+/+ oocytes.Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.

Tau-mediated nuclear depletion and cytoplasmic accumulation of SFPQ in Alzheimer's and Pick's disease.

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Yazi D Ke

Full Text Available Tau dysfunction characterizes neurodegenerative diseases such as Alzheimer's disease (AD and frontotemporal lobar degeneration (FTLD. Here, we performed an unbiased SAGE (serial analysis of gene expression of differentially expressed mRNAs in the amygdala of transgenic pR5 mice that express human tau carrying the P301L mutation previously identified in familial cases of FTLD. SAGE identified 29 deregulated transcripts including Sfpq that encodes a nuclear factor implicated in the splicing and regulation of gene expression. To assess the relevance for human disease we analyzed brains from AD, Pick's disease (PiD, a form of FTLD, and control cases. Strikingly, in AD and PiD, both dementias with a tau pathology, affected brain areas showed a virtually complete nuclear depletion of SFPQ in both neurons and astrocytes, along with cytoplasmic accumulation. Accordingly, neurons harboring either AD tangles or Pick bodies were also depleted of SFPQ. Immunoblot analysis of human entorhinal cortex samples revealed reduced SFPQ levels with advanced Braak stages suggesting that the SFPQ pathology may progress together with the tau pathology in AD. To determine a causal role for tau, we stably expressed both wild-type and P301L human tau in human SH-SY5Y neuroblastoma cells, an established cell culture model of tau pathology. The cells were differentiated by two independent methods, mitomycin C-mediated cell cycle arrest or neuronal differentiation with retinoic acid. Confocal microscopy revealed that SFPQ was confined to nuclei in non-transfected wild-type cells, whereas in wild-type and P301L tau over-expressing cells, irrespective of the differentiation method, it formed aggregates in the cytoplasm, suggesting that pathogenic tau drives SFPQ pathology in post-mitotic cells. Our findings add SFPQ to a growing list of transcription factors with an altered nucleo-cytoplasmic distribution under neurodegenerative conditions.

Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells

International Nuclear Information System (INIS)

Duncan, L; Shelmerdine, H; Hughes, M P; Coley, H M; Huebner, Y; Labeed, F H

2008-01-01

Dielectrophoresis (DEP)-the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K + and Ca 2+ levels were extremely similar between sensitive and resistant lines, levels of Cl - were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function. (note)

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

Science.gov (United States)

Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

2014-04-14

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

Science.gov (United States)

Favini-Stabile, Sandy; Contreras-Martel, Carlos; Thielens, Nicole; Dessen, Andréa

2013-12-01

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Plasma exchange and glucocorticoid dosing in the treatment of anti-neutrophil cytoplasm antibody associated vasculitis (PEXIVAS)

DEFF Research Database (Denmark)

Walsh, Michael; Merkel, Peter A; Peh, Chen Au

2013-01-01

Granulomatosis with polyangiitis (GPA, Wegener's) and microscopic polyangiitis (MPA) are small vessel vasculitides collectively referred to as anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). AAV is associated with high rates of morbidity and mortality due to uncontrolled disease...

[Antineutrophil cytoplasmic antibodies associated with infective endocarditis: Literature review].

Science.gov (United States)

Langlois, V; Marie, I

2017-07-01

Antineutrophil cytoplasmic antibodies (ANCA) associated with infective endocarditis are a rare disorder. The condition can mimic primary systemic vasculitis (i.e. granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis). Thus, a wrong diagnosis of valvular involvement related to primary systemic vasculitis can be made in patients exhibiting ANCA associated with infective endocarditis. Because treatment of both conditions is different, this wrong diagnosis will lead to dramatic consequences in these latter patients. This review reports the state of knowledge and proposes an algorithm to follow when confronted to a possible case of ANCA associated with infective endocarditis. Copyright © 2016 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier SAS. All rights reserved.

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

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Shimura Takaya

2012-05-01

Full Text Available Abstract Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C, translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF and mutated HB-EGF (HB-EGF-mC, which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 % and in the cytoplasm only in 25 cases (26.0 %. The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P  Conclusions Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation might be crucial in gastric cancer invasion. HB-EGF-C nuclear translocation may offer a prognostic marker and a new molecular target for gastric cancer therapy.

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea

NARCIS (Netherlands)

van de Vossenberg, J.L C M; Ubbink-Kok, T.; Elferink, M.G.L.; Driessen, A.J.M.; Konings, W.N

1995-01-01

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is

Development of new semidwarf sources for rice with different cytoplasms (CV Basmati 370 and Gloria)

Energy Technology Data Exchange (ETDEWEB)

Deus, J E; Suarez, E [Instituto de Investigaciones del Arroz, Bauta, Havana (Cuba); Fuentes, J L; Alvarez, A; Padron, E [Centro de Estudios Aplicados al Desarrollo Nuclear, Miramar, Playa, Havana (Cuba)

2001-05-01

In Cuba semidwarf rice varieties grow on 98% of the area. Virtually all carry the same Dee-geo-won-gen dwarfing gene. Also, most if not all the cultivars have the same cytoplasm. The induced mutations approach was undertaken in order to generate alternative genetic sources of dwarfing with different cytoplasms and to improve the grain quality of Cuban rice. The seeds of two varieties, Basmati 370 and Gloria, were irradiated with 200 and 300 Gy of {sup 60}Co gamma rays. In several generations of selection, progeny testing and preliminary yield tests 10 mutants (6 from Basmati 370 and 4 from Gloria), whose yields exceeded the source cultivars, have been advanced to replicated yield trials. Some of the mutant lines are also resistant to lodging and to blast (Pyricularis grisea). It is concluded the induced mutations can be used successfully to improve plant type and other agronomic traits in rice. The induced mutants will be used also in hybridization programs. (author)

Development of new semidwarf sources for rice with different cytoplasms (CV Basmati 370 and Gloria)

International Nuclear Information System (INIS)

Deus, J.E.; Suarez, E.; Fuentes, J.L.; Alvarez, A.; Padron, E.

2001-01-01

In Cuba semidwarf rice varieties grow on 98% of the area. Virtually all carry the same Dee-geo-won-gen dwarfing gene. Also, most if not all the cultivars have the same cytoplasm. The induced mutations approach was undertaken in order to generate alternative genetic sources of dwarfing with different cytoplasms and to improve the grain quality of Cuban rice. The seeds of two varieties, Basmati 370 and Gloria, were irradiated with 200 and 300 Gy of 60 Co gamma rays. In several generations of selection, progeny testing and preliminary yield tests 10 mutants (6 from Basmati 370 and 4 from Gloria), whose yields exceeded the source cultivars, have been advanced to replicated yield trials. Some of the mutant lines are also resistant to lodging and to blast (Pyricularis grisea). It is concluded the induced mutations can be used successfully to improve plant type and other agronomic traits in rice. The induced mutants will be used also in hybridization programs. (author)

Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

Science.gov (United States)

Chen, Tai-Yen; Jung, Won; Santiago, Ace George; Yang, Feng; Krzemiński, Łukasz; Chen, Peng

2015-11-12

Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

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Lobstein Julie

2012-05-01

Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

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Cecilia Zampedri

2016-10-01

Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

Science.gov (United States)

Bevacqua, R. J.; Fernandez-Martin, R.; Canel, N. G.; Gibbons, A.; Texeira, D.; Lange, F.; Vans Landschoot, G.; Savy, V.; Briski, O.; Hiriart, M. I.; Grueso, E.; Ivics, Z.; Taboga, O.; Kues, W. A.; Ferraris, S.

2017-01-01

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest. PMID:28301581

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

Science.gov (United States)

Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

2005-10-20

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Regulation of autophagy by cytoplasmic p53.

Science.gov (United States)

Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

2008-06-01

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Association of nad7a Gene with Cytoplasmic Male Sterility in Pigeonpea

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Pallavi Sinha

2015-07-01

Full Text Available Cytoplasmic male sterility (CMS has been exploited in the commercial pigeonpea [ (L. Millsp.] hybrid breeding system; however, the molecular mechanism behind this system is unknown. To understand the underlying molecular mechanism involved in A CMS system derived from (Haines Maesen, 34 mitochondrial genes were analyzed for expression profiling and structural variation analysis between CMS line (ICRISAT Pigeonpea A line, ICPA 2039 and its cognate maintainer (ICPB 2039. Expression profiling of 34 mitochondrial genes revealed nine genes with significant fold differential gene expression at ≤ 0.01, including one gene, , with 1366-fold higher expression in CMS line as compared with the maintainer. Structural variation analysis of these mitochondrial genes identified length variation between ICPA 2039 and ICPB 2039 for (subunit of gene. Sanger sequencing of and genes in the CMS and the maintainer lines identified two single nucleotide polymorphisms (SNPs in upstream region of and a deletion of 10 bp in in the CMS line. Protein structure evaluation showed conformational changes in predicted protein structures for between ICPA 2039 and ICPB 2039 lines. All above analyses indicate association of gene with the CMS for A cytoplasm in pigeonpea. Additionally, one polymerase chain reaction (PCR based Indel marker ( has been developed and validated for testing genetic purity of A derived CMS lines to strengthen the commercial hybrid breeding program in pigeonpea.

The effect of the external medium on the gravity-induced polarity of cytoplasmic streaming in Chara corallina (Characeae)

Science.gov (United States)

Staves, M. P.; Wayne, R.; Leopold, A. C.

1997-01-01

Gravity induces a polarity of cytoplasmic streaming in vertical internodal cells of Chara such that the downwardly directed stream moves faster than the upwardly directed stream. In order to determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best explain the gravity response in Chara internodal cells, we controlled the physical properties of the external medium, including density and osmolarity, with impermeant solutes and examined the effect on the polarity of cytoplasmic streaming. As the density of the external medium is increased, the polarity of cytoplasmic streaming decreases and finally disappears when the density of the external medium is equal to that of the cell (1015 kg/m3). A further increase in the density of the external medium causes a reversal of the gravity response. These results are consistent with the gravitational pressure theory of gravity sensing since the buoyancy of the protoplast is dependent on the difference between the density of the protoplast and the external medium, and are inconsistent with the statolith theory since the buoyancy of intracellular particles are unaffected by changes in the external medium.

Evidence for significantly enhancing reduction of Azo dyes in Escherichia coli by expressed cytoplasmic Azoreductase (AzoA) of Enterococcus faecalis.

Science.gov (United States)

Feng, J; Heinze, T M; Xu, H; Cerniglia, C E; Chen, H

2010-05-01

Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

Effects of chromosomal breaks induced by X-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

International Nuclear Information System (INIS)

Parks, L.C.; Dicker, D.T.; Conger, A.D.; Daneo-Moore, L.; Higgins, M.L.

1981-01-01

A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by 1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; 2) subsequent determination of the number of irradiated cells in which mesosomes and central density changes could be visualised after fixative was added. The results obtained by exposure of cells to a) doses up to 1100 krads before fixation and b) doses greater than 1100 krads before fixation suggested that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted. (author)

Analysis of embryo, cytoplasm and maternal effects on fatty acid components in soybean (Glycine max Merill.)

Institute of Scientific and Technical Information of China (English)

NING Hailong; LI Wenxia; LI Wenbin

2007-01-01

The quality of oil determined by the constituents and proportion of fatty acid components,and the understanding of heredity of fatty acid components are of importance to breeding good quality soybean varieties.Embryo,cytoplasmic and maternal effects and genotype×environment interaction effects for quality traits of soybean [Glycine max (L.) Merrill.] seeds were analyzed using a general genetic model for quantitative traits of seeds with parents,F1 and F2,of 20 crosses from a diallel mating design of five parents planted in the field in 2003 and 2004 in Harbin,China.The interaction effects of palmitic,stearic,and linoleic acid contents were larger than the genetic main effects,while the genetic main effects were equal to interaction effects for linolenic and oleic acid content.Among all kinds of genetic main effects,the embryo effects were the largest for palmitic,stearic,and linoleic acids,while the cytoplasm effects were the largest for oleic and linolenic acids.Among all kinds of interaction effects,the embryo interaction effects were the largest for fatty acids.The sum of additive and additive× environment effects were larger than that of dominance and dominance×environment effects for the linolenic acid content,but not for other quality traits.The general heritabilities were the main parts of heritabilities for palmitic and oleic acid contents,but the interaction was more important for stearic,linoleic,and linolenic acid contents.For the general heritability,maternal and cytoplasm heritabilities were the main components for palmitic,oleic,and linolenic acid contents.It was shown for the interaction heritabilities that the embryo interaction heritabilities were more important for oleic and linolenic acid contents,while the maternal interaction heritabilities were more important for linoleic acid content.Among selection response components,the maternal and cytoplasm general responses and/or interaction responses were more important for palmitic

Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen.

NARCIS (Netherlands)

Motta, A C; Marliere, M; Peltre, G; Sterenberg, P A; Lacroix, G

2006-01-01

BACKGROUND/AIM: Pollen cytoplasmic granules (PCG) are loaded with allergens. They are released from grass pollen grains following contact with water and can form a respirable allergenic aerosol. On the other hand, the traffic-related air pollutants NO2 and O3 are known to be involved in the current

Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen

NARCIS (Netherlands)

Motta, AC; Marliere, M; Peltre, G; Sterenberg, PA; Lacroix, G

2006-01-01

Background/Aim: Pollen cytoplasmic granules (PCG) are loaded with allergens. They are released from grass pollen grains following contact with water and can form a respirable allergenic aerosol. On the other hand, the traffic-related air pollutants NO2 and O-3 are known to be involved in the current

Solution structure of a syndecan-4 cytoplasmic domain and its interaction with phosphatidylinositol 4,5-bisphosphate

DEFF Research Database (Denmark)

Lee, D; Oh, E S; Woods, A

1998-01-01

Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Calpha and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V......) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data...... and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits...

Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

Science.gov (United States)

Azumaya, Caleigh M; Sierra-Valdez, Francisco; Cordero-Morales, Julio F; Nakagawa, Terunaga

2018-05-11

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here we report a cryoEM structure of the cytoplasmic domain of murine TRPC6 at 3.8Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike in other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoplasmic inheritance of parent-offspring cell structure in the clonal diatom Cyclotella meneghiniana.

Science.gov (United States)

Shirokawa, Yuka; Shimada, Masakazu

2016-11-16

In cytoplasmic inheritance, structural states of a parent cell could be transmitted to offspring cells via two mechanisms. The first is referred to as the hangover of parent structure, where the structure itself remains and faithfully transmits within offspring cells; the second is structural inheritance, wherein the parent structure functions as a template for development of new offspring structure. We estimated to what extent the parent structure affects the development of offspring structure by structural inheritance, using a clone of the diatom Cyclotella meneghiniana The cell has two siliceous valves (a cell wall part at both cell poles): one is inherited from the parent and the other is newly formed. We estimated cytoplasmic heritability by comparing valve traits (central fultoportulae (CTFP), striae, central area, and cell diameter) of parent and new offspring valves, using single-cell isolation and valve labelling. Parent-offspring valve trait regressions showed that all traits, except CTFP, were significantly correlated. We formulated a quantitative genetic model considering the diatom inheritance system and revealed short-term rapid evolution compared with other inheritance systems. Diatom structural inheritance will have evolved to enable clonal populations to rapidly acquire and maintain suitable structures for temporal changes in environments and life-cycle stages. © 2016 The Author(s).

The RanGTP pathway: from nucleo-cytoplasmic transport to spindle assembly and beyond

Directory of Open Access Journals (Sweden)

Tommaso eCavazza

2016-01-01

Full Text Available The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context.

Interferometric measurements of dry mass content in nuclei and cytoplasm in the life cycle of antheridial filaments cells of Chara vulgaris L. in their successive developmental stages

Directory of Open Access Journals (Sweden)

Hanna Kuran

2015-01-01

Full Text Available Interferometric measurements of the nucleus and cytoplasm dry mass during interphase in the successive stages of development of antheridial filaments of Chara vulgaris demonstrated that the dry mass and surface area of cell nuclei double in size in each of the successive generations of the filaments, whereas neither the surface nor the dry mass of the cytoplasm increase in such proportion in the same period. In the successive stages of development of the antheridial filaments the dry mass and surface area of the nuclei and cytoplasm gradually diminish.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

Science.gov (United States)

Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

2015-01-01

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets

Science.gov (United States)

Noujaim, Sami F.; Stuckey, Jeanne A.; Ponce-Balbuena, Daniela; Ferrer-Villada, Tania; López-Izquierdo, Angelica; Pandit, Sandeep; Calvo, Conrado J.; Grzeda, Krzysztof R.; Berenfeld, Omer; Sánchez Chapula, José A.; Jalife, José

2010-01-01

Atrial and ventricular tachyarrhythmias can be perpetuated by up-regulation of inward rectifier potassium channels. Thus, it may be beneficial to block inward rectifier channels under conditions in which their function becomes arrhythmogenic (e.g., inherited gain-of-function mutation channelopathies, ischemia, and chronic and vagally mediated atrial fibrillation). We hypothesize that the antimalarial quinoline chloroquine exerts potent antiarrhythmic effects by interacting with the cytoplasmic domains of Kir2.1 (IK1), Kir3.1 (IKACh), or Kir6.2 (IKATP) and reducing inward rectifier potassium currents. In isolated hearts of three different mammalian species, intracoronary chloroquine perfusion reduced fibrillatory frequency (atrial or ventricular), and effectively terminated the arrhythmia with resumption of sinus rhythm. In patch-clamp experiments chloroquine blocked IK1, IKACh, and IKATP. Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of Kir2.1, Kir3.1, and Kir6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. These results open a novel path toward discovering antiarrhythmic pharmacophores that target specific residues of the cytoplasmic domain of inward rectifier potassium channels.—Noujaim, S. F., Stuckey, J. A., Ponce-Balbuena, D., Ferrer-Villada, T., López-Izquierdo, A., Pandit, S., Calvo, C. J., Grzeda, K. R., Berenfeld, O., Sánchez Chapula, J. A., Jalife, J. Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets. PMID:20585026

Direct Cytoplasmic Delivery and Nuclear Targeting Delivery of HPMA-MT Conjugates in a Microtubules Dependent Fashion.

Science.gov (United States)

Zhong, Jiaju; Zhu, Xi; Luo, Kui; Li, Lian; Tang, Manlin; Liu, Yanxi; Zhou, Zhou; Huang, Yuan

2016-09-06

As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve direct cytoplasmic delivery by a nonendocytic pathway and transport into the nucleus in a microtubules dependent fashion. A special targeting peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into the nucleus a by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by a nonendocytic pathway, thus avoiding the drug degradation in the lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the DTX and Noc treated groups. These results demonstrated that the conjugates transported into the nucleus in a microtubules dependent way. Therefore, in addition to direct cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

A novel mitochondrial orf147 causes cytoplasmic male sterility in pigeonpea by modulating aberrant anther dehiscence.

Science.gov (United States)

Bhatnagar-Mathur, Pooja; Gupta, Ranadheer; Reddy, Palakolanu Sudhakar; Reddy, Bommineni Pradeep; Reddy, Dumbala Srinivas; Sameerkumar, C V; Saxena, Rachit Kumar; Sharma, Kiran K

2018-05-01

A novel open reading frame (ORF) identified and cloned from the A4 cytoplasm of Cajanus cajanifolius induced partial to complete male sterility when introduced into Arabidopsis and tobacco. Pigeonpea (Cajanus cajan L. Millsp.) is the only legume known to have commercial hybrid seed technology based on cytoplasmic male sterility (CMS). We identified a novel ORF (orf147) from the A4 cytoplasm of C. cajanifolius that was created via rearrangements in the CMS line and co-transcribes with the known and unknown sequences. The bi/poly-cistronic transcripts cause gain-of-function variants in the mitochondrial genome of CMS pigeonpea lines having distinct processing mechanisms and transcription start sites. In presence of orf147, significant repression of Escherichia coli growth indicated its toxicity to the host cells and induced partial to complete male sterility in transgenic progenies of Arabidopsis thaliana and Nicotiana tabacum where phenotype co-segregated with the transgene. The male sterile plants showed aberrant floral development and reduced lignin content in the anthers. Gene expression studies in male sterile pigeonpea, Arabidopsis and tobacco plants confirmed down-regulation of several anther biogenesis genes and key genes involved in monolignol biosynthesis, indicative of regulation of retrograde signaling. Besides providing evidence for the involvement of orf147 in pigeonpea CMS, this study provides valuable insights into its function. Cytotoxicity and aberrant programmed cell death induced by orf147 could be important for mechanism underlying male sterility that offers opportunities for possible translation for these findings for exploiting hybrid vigor in other recalcitrant crops as well.

Novel CMS lines in pigeonpea [Cajanus cajan (L. Millspaugh] derived from cytoplasmic substitutions, and their effective restoration and deployment in hybrid breeding

Directory of Open Access Journals (Sweden)

Abhishek Bohra

2017-02-01

Full Text Available The availability of stable cytoplasmic male sterile (CMS or A lines coupled with a robust restoration system (R lines is an essential prerequisite for efficient hybrid breeding. CMS-enabled hybrid technology holds immense potential to enhance the long-stagnant productivity of pigeonpea. In the present investigation, cytoplasmic substitutions were made in the nuclear backgrounds of early-maturing pigeonpea varieties or lines. Three new CMS lines (ICPL 88039A, Pusa 992A, and DPP 3-2A resulted from genetic crosses involving cytoplasmic donors from A2 (GT 288A and A4 (ICPA 2089 categories. In addition to visual inspection of anthers, pollen-staining techniques and scanning electron microscopy (SEM analysis were used to confirm pollen sterility. Further, given the relevance of the plant mitochondrial genome to CMS manifestation, 25 mitochondrion-specific DNA markers were assayed on these newly developed A lines and isogenic maintainer (B lines. DNA polymorphism between Pusa 992A and Pusa 992B as revealed by the nad7a_del marker confirmed the successful combination of sterilizing cytoplasm (A4 and nonrestoring nuclear background (Pusa 992. Such cytoplasm-specific DNA markers are required for A2-CMS as well. Further, to assess restoration ability, potential restorers were crossed with these CMS lines, and as a consequence, promising A × R combinations exhibiting 100% pollen fertility could be identified. In parallel, we also analyzed the inheritance patterns underlying fertility restoration using ICPL 88039A-derived F2 and BC1F1 populations, and established a monogenic dominant model to explain the phenomenon of A2-CMS restoration. In summary, we report the successful development of new CMS lines and describe their effective deployment in hybrid breeding of pigeonpea.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

Science.gov (United States)

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

Directory of Open Access Journals (Sweden)

Deepshi Thakral

Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

Positive Selection Driving Cytoplasmic Genome Evolution of the Medicinally Important Ginseng Plant Genus Panax.

Science.gov (United States)

Jiang, Peng; Shi, Feng-Xue; Li, Ming-Rui; Liu, Bao; Wen, Jun; Xiao, Hong-Xing; Li, Lin-Feng

2018-01-01

Panax L. (the ginseng genus) is a shade-demanding group within the family Araliaceae and all of its species are of crucial significance in traditional Chinese medicine. Phylogenetic and biogeographic analyses demonstrated that two rounds of whole genome duplications accompanying with geographic and ecological isolations promoted the diversification of Panax species. However, contributions of the cytoplasmic genomes to the adaptive evolution of Panax species remained largely uninvestigated. In this study, we sequenced the chloroplast and mitochondrial genomes of 11 accessions belonging to seven Panax species. Our results show that heterogeneity in nucleotide substitution rate is abundant in both of the two cytoplasmic genomes, with the mitochondrial genome possessing more variants at the total level but the chloroplast showing higher sequence polymorphisms at the genic regions. Genome-wide scanning of positive selection identified five and 12 genes from the chloroplast and mitochondrial genomes, respectively. Functional analyses further revealed that these selected genes play important roles in plant development, cellular metabolism and adaptation. We therefore conclude that positive selection might be one of the potential evolutionary forces that shaped nucleotide variation pattern of these Panax species. In particular, the mitochondrial genes evolved under stronger selective pressure compared to the chloroplast genes.

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

Science.gov (United States)

Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke; Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho; Yamamoto, Akitsugu; Nakamura, Nobuhiro

2014-11-01

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

Science.gov (United States)

Girard, Brian J; Regan Anderson, Tarah M; Welch, Siya Lem; Nicely, Julie; Seewaldt, Victoria L; Ostrander, Julie H

2015-01-01

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

Making it big : how characean algae use cytoplasmic streaming to enhance transport in giant cells

NARCIS (Netherlands)

Meent, Jan Willem van de

2010-01-01

Organisms show a remarkable variation in sizes, yet cell sizes are surprisingly similar across species, typically ranging from 10 μm to 100 μm. A striking exception are the giant cells of the algal weed Chara, which can exceed 10 cm in length and 1 mm in diameter. A circulation known as cytoplasmic

The early noncoding region of human papillomavirus type 16 is regulated by cytoplasmic polyadenylation factors

DEFF Research Database (Denmark)

Glahder, Jacob-Andreas Harald; Kristiansen, Karen; Durand, Marjorie

2010-01-01

All human papillomavirus type 16 (HPV-16) early mRNAs are polyadenylated at the poly(A) signal within the early 3' untranslated region (3'UTR). The 3'end of the early E5 open reading frame and the 3'UTR of HPV-16 is very AU-rich, with five regions similar to cytoplasmic polyadenylation elements (...

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

Science.gov (United States)

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds

NARCIS (Netherlands)

Buitink, J.; Leprince, O.; Hoekstra, F.A.

2000-01-01

This study establishes a relationship between desiccation tolerance and the transfer of amphiphilic molecules from the cytoplasm into lipids during drying, using electron paramagnetic resonance spectroscopy of amphiphilic spin probes introduced into imbibed radicles of pea (Pisum sativum) and

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

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Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

2014-10-15

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Perez-Ramirez, B.; Martinez-Carrion, M.

1989-01-01

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

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Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

2012-05-30

Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation

Urinary CD4+ Effector Memory T Cells Reflect Renal Disease Activity in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

NARCIS (Netherlands)

Abdulahad, Wayel H.; Kallenberg, Cees G. M.; Limburg, Pieter C.; Stegeman, Coen A.

Objective. Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration

Confocal Microscopy and Image Analysis Indicates a Region-Specific Relation between Active Caspases and Cytoplasm in Ejaculated and Epididymal Sperm

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García Vazquez, Susana; Aragón Martínez, Andrés; Flores-Alonso, Juan Carlos

2012-01-01

Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm. PMID:22530029

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors

International Nuclear Information System (INIS)

Pozsgai, Eva; Gomori, Eva; Szigeti, Andras; Boronkai, Arpad; Gallyas, Ferenc Jr; Sumegi, Balazs; Bellyei, Szabolcs

2007-01-01

Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. Low grade (grades 1–2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker

Validation of cytoplasmic-to-nuclear ratio of survivin as an indicator of improved prognosis in breast cancer

International Nuclear Information System (INIS)

Rexhepaj, Elton; Jirstrom, Karin; O'Connor, Darran P; O'Brien, Sallyann L; Landberg, Goran; Duffy, Michael J; Brennan, Donal J; Gallagher, William M

2010-01-01

Conflicting data exist regarding the prognostic and predictive impact of survivin (BIRC5) in breast cancer. We previously reported survivin cytoplasmic-to-nuclear ratio (CNR) as an independent prognostic indicator in breast cancer. Here, we validate survivin CNR in a separate and extended cohort. Furthermore, we present new data suggesting that a low CNR may predict outcome in tamoxifen-treated patients. Survin expression was assessed using immunhistochemistry on a breast cancer tissue microarray (TMA) containing 512 tumours. Whole slide digital images were captured using an Aperio XT scanner. Automated image analysis was used to identify tumour from stroma and then to quantify tumour-specific nuclear and cytoplasmic survivin. A decision tree model selected using a 10-fold cross-validation approach was used to identify prognostic subgroups based on nuclear and cytoplasmic survivin expression. Following optimisation of the staining procedure, it was possible to evaluate survivin protein expression in 70.1% (n = 359) of the 512 tumours represented on the TMA. Decision tree analysis predicted that nuclear, as opposed to cytoplasmic, survivin was the most important determinant of overall survival (OS) and breast cancer-specific survival (BCSS). The decision tree model confirmed CNR of 5 as the optimum threshold for survival analysis. Univariate analysis demonstrated an association between a high CNR (>5) and a prolonged BCSS (HR 0.49, 95% CI 0.29-0.81, p = 0.006). Multivariate analysis revealed a high CNR (>5) was an independent predictor of BCSS (HR 0.47, 95% CI 0.27-0.82, p = 0.008). An increased CNR was associated with ER positive (p = 0.045), low grade (p = 0.007), Ki-67 (p = 0.001) and Her2 (p = 0.026) negative tumours. Finally, a high CNR was an independent predictor of OS in tamoxifen-treated ER-positive patients (HR 0.44, 95% CI 0.23-0.87, p = 0.018). Using the same threshold as our previous study, we have validated survivin CNR as a marker of good prognosis in

Validation of cytoplasmic-to-nuclear ratio of survivin as an indicator of improved prognosis in breast cancer

LENUS (Irish Health Repository)

Rexhepaj, Elton

2010-11-23

Abstract Background Conflicting data exist regarding the prognostic and predictive impact of survivin (BIRC5) in breast cancer. We previously reported survivin cytoplasmic-to-nuclear ratio (CNR) as an independent prognostic indicator in breast cancer. Here, we validate survivin CNR in a separate and extended cohort. Furthermore, we present new data suggesting that a low CNR may predict outcome in tamoxifen-treated patients. Methods Survin expression was assessed using immunhistochemistry on a breast cancer tissue microarray (TMA) containing 512 tumours. Whole slide digital images were captured using an Aperio XT scanner. Automated image analysis was used to identify tumour from stroma and then to quantify tumour-specific nuclear and cytoplasmic survivin. A decision tree model selected using a 10-fold cross-validation approach was used to identify prognostic subgroups based on nuclear and cytoplasmic survivin expression. Results Following optimisation of the staining procedure, it was possible to evaluate survivin protein expression in 70.1% (n = 359) of the 512 tumours represented on the TMA. Decision tree analysis predicted that nuclear, as opposed to cytoplasmic, survivin was the most important determinant of overall survival (OS) and breast cancer-specific survival (BCSS). The decision tree model confirmed CNR of 5 as the optimum threshold for survival analysis. Univariate analysis demonstrated an association between a high CNR (>5) and a prolonged BCSS (HR 0.49, 95% CI 0.29-0.81, p = 0.006). Multivariate analysis revealed a high CNR (>5) was an independent predictor of BCSS (HR 0.47, 95% CI 0.27-0.82, p = 0.008). An increased CNR was associated with ER positive (p = 0.045), low grade (p = 0.007), Ki-67 (p = 0.001) and Her2 (p = 0.026) negative tumours. Finally, a high CNR was an independent predictor of OS in tamoxifen-treated ER-positive patients (HR 0.44, 95% CI 0.23-0.87, p = 0.018). Conclusion Using the same threshold as our previous study, we have

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.

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Tanaka Yoshiyuki

2012-07-01

Full Text Available Abstract Background Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. Results Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%. It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size that are non-syntenic to normal-type genome, and the gene orf138

Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

NARCIS (Netherlands)

Long, G.; Pan, M.; Westenberg, M.; Vlak, J.M.

2006-01-01

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD),

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

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Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

2015-02-18

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytoplasmic localization of Hug1p, a negative regulator of the MEC1 pathway, coincides with the compartmentalization of Rnr2p–Rnr4p

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Ainsworth, William B. [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Hughes, Bridget Todd; Au, Wei Chun; Sakelaris, Sally [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Kerscher, Oliver [Biology Department, The College of William and Mary, Williamsburg, VA 23185 (United States); Benton, Michael G., E-mail: benton@lsu.edu [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Basrai, Munira A., E-mail: basraim@mail.nih.gov [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

2013-10-04

Highlights: •Hug1p overexpression sensitizes wild-type cells to DNA damage and hydroxyurea (HU). •Expression of Hug1p in response to HU treatment is delayed relative to Rnr3p. •MEC1 pathway genes are required for cytoplasmic localization of Hug1p. •Hug1p subcellular compartmentalization to the cytoplasm coincides with Rnr2p–Rnr4p. -- Abstract: The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p–Rnr4p subunits and inactivation of negative regulators Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p–Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p–Rnr4p.

Higher cytoplasmic and nuclear poly(ADP-ribose) polymerase expression in familial than in sporadic breast cancer

NARCIS (Netherlands)

Klauke, M.L.; Hoogerbrugge-van der Linden, N.; Budczies, J.; Bult, P.; Prinzler, J.; Radke, C.; van Krieken, J.H.; Dietel, M.; Denkert, C.; Muller, B.M.

2012-01-01

Poly(ADP-ribose) polymerase 1 (PARP) is a key element of the single-base excision pathway for repair of DNA single-strand breaks. To compare the cytoplasmic and nuclear poly(ADP-ribose) expression between familial (BRCA1, BRCA2, or non BRCA1/2) and sporadic breast cancer, we investigated 39 sporadic

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

Science.gov (United States)

Banks, Gareth T; Haas, Matilda A; Line, Samantha; Shepherd, Hazel L; Alqatari, Mona; Stewart, Sammy; Rishal, Ida; Philpott, Amelia; Kalmar, Bernadett; Kuta, Anna; Groves, Michael; Parkinson, Nicholas; Acevedo-Arozena, Abraham; Brandner, Sebastian; Bannerman, David; Greensmith, Linda; Hafezparast, Majid; Koltzenburg, Martin; Deacon, Robert; Fainzilber, Mike; Fisher, Elizabeth M C

2011-04-06

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

OpenAIRE

Puddington, L; Woodgett, C; Rose, J K

1987-01-01

The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

Science.gov (United States)

Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

2017-01-01

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Science.gov (United States)

Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

2015-05-01

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Directory of Open Access Journals (Sweden)

Alene Kast

2015-05-01

Full Text Available Cytoplasmic virus like elements (VLEs from Kluyveromyces lactis (Kl, Pichia acaciae (Pa and Debaryomyces robertsiae (Dr are extremely A/T-rich (>75% and encode toxic anticodon nucleases (ACNases along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5 results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2 and malignancy in brain tumors

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Gallyas Ferenc

2007-12-01

Full Text Available Abstract Background Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. Methods Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+, moderate (++, high (+++ or none (- scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. Results Low grade (grades 1–2 brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4 tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. Conclusion Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane.

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Kumari, Nimerta; Götz, Friedrich; Nguyen, Minh-Thu

2017-12-01

In gram-negative bacteria, the ABC transporter LolCDE complex translocates outer membrane-specific lipoproteins (Lpp) from the inner membrane to the outer membrane. Lpp possessing aspartate (Asp) at position +2 are not translocated because it functions as a LolCDE avoidance signal. In gram-positive bacteria, lacking an outer membrane and the Lol system, Lpp are only anchored at the outer leaflet of the cytoplasmic membrane. However, the release of Lpp particularly in pathogenic or commensal species is crucial for immune modulation. Here, we provide evidence that in Staphylococcus aureus Asp at position +2 plays a role in withholding Lpp to the cytoplasmic membrane. Screening of published exoproteomic data of S. aureus revealed that Lpp mainly with Gly or Ser at position +2 were found in exoproteome, but there was no Lpp with Asp+2. The occurrence of Lpp with Asp+2 is infrequent in gram-positive bacteria. In S. aureus USA300 only seven of the 67 Lpp possess Asp+2; among them five Lpp represented Lpl lipoproteins involved in host cell invasion. Our study demonstrated that replacing the Asp+2 present in Lpl8 with a Ser enhances its release into the supernatant. However, there is no different release of Asp+2 and Ser+2 in mprF mutant that lacks the positive charge of lysyl-phosphatidylglycerol (Lys-PG). Moreover, substitution of Ser+2 by Asp in SitC (MntC) did not lead to a decreased release indicating that in staphylococci positions +3 and +4 might also be important for a tighter anchoring of Lpp. Here, we show that Asp in position +2 and adjacent amino acids contribute in tightening the anchoring of Lpp by interaction of the negative charged Asp with the positive charged Lys-PG. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes.

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Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-09-14

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes.

Reduced RAN expression and disrupted transport between cytoplasm and nucleus; a key event in Alzheimer's disease pathophysiology.

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Diego Mastroeni

Full Text Available Transcription of DNA is essential for cell maintenance and survival; inappropriate localization of proteins that are involved in transcription would be catastrophic. In Alzheimer's disease brains, and in vitro studies, we have found qualitative and quantitative deficits in transport into the nucleus of DNA methyltransferase 1 (DNMT1 and RNA polymerase II (RNA pol II, accompanied by their abnormal sequestration in the cytoplasm. RAN (RAs-related Nuclear protein knockdown, by siRNA and oligomeric Aβ42 treatment in neurons, replicate human data which indicate that transport disruption in AD may be mechanistically linked to reduced expression of RAN, a pivotal molecule in nucleocytoplasmic transport. In vitro studies also indicate a significant role for oligomeric Aβ42 in the observed phenomena. We propose a model in which reduced transcription regulators in the nucleus and their increased presence in the cytoplasm may lead to many of the cellular manifestations of Alzheimer's disease.

Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

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Christos Tzitzilonis

Full Text Available Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15N,(1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15N,(1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

International Nuclear Information System (INIS)

Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G.

2006-01-01

Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites

Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

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Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

2010-08-06

The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

Investigation of radiation enhanced reactivation of cytoplasmic replicating human virus

International Nuclear Information System (INIS)

Bockstahler, L.E.; Haynes, K.F.; Stafford, J.E.

1976-01-01

When monolayers of CV-1 monkey kidney cells were exposed to ultraviolet (uv) radiation (0 to 200 erg/nm 2 ) or x rays (0 to 10 krads) before infection with uv-irradiated herpes simplex virus, an increase in the infectivity of this nuclear replicating virus occurred as measured by plaque formation. These phenomena are known as uv (Weigle) reactivation (WR) and x-ray reactivation (x-ray R). In this study the presence of WR and x-ray R was examined in CV-1 cells infected with uv-irradiated vaccinia virus or poliovirus, both cytoplasmic replicating viruses. Little or no WR or x-ray R was observed for either of these viruses. These results suggest that WR and x-ray R in mammalian cells may be restricted to viruses which are synthesized in the cell nucleus

Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.).

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Guo, Jinju; Wang, Peng; Cheng, Qing; Sun, Limin; Wang, Hongyu; Wang, Yutong; Kao, Lina; Li, Yanan; Qiu, Tuoyu; Yang, Wencai; Shen, Huolin

2017-09-25

Although cytoplasmic male sterility (CMS) is widely used for developing pepper hybrids, its molecular mechanism remains unclear. In this study, we used a high-throughput proteomics method called label-free to compare protein abundance across a pepper CMS line (A-line) and its isogenic maintainer line (B-line). Data are available via ProteomeXchange with identifier PXD006104. Approximately 324 differentially abundant protein species were identified and quantified; among which, 47 were up-accumulated and 140 were down-accumulated in the A-line; additionally, 75 and 62 protein species were specifically accumulated in the A-line and B-line, respectively. Protein species involved in pollen exine formation, pyruvate metabolic processes, the tricarboxylic acid cycle, the mitochondrial electron transport chain, and oxidative stress response were observed to be differentially accumulated between A-line and B-line, suggesting their potential roles in the regulation of pepper pollen abortion. Based on our data, we proposed a potential regulatory network for pepper CMS that unifies these processes. Artificial emasculation is a major obstacle in pepper hybrid breeding for its high labor cost and poor seed purity. While the use of cytoplasmic male sterility (CMS) in hybrid system is seriously frustrated because a long time is needed to cultivate male sterility line and its isogenic restore line. Transgenic technology is an effective and rapid method to obtain male sterility lines and its widely application has very important significance in speeding up breeding process in pepper. Although numerous studies have been conducted to select the genes related to male sterility, the molecular mechanism of cytoplasmic male sterility in pepper remains unknown. In this study, we used the high-throughput proteomic method called "label-free", coupled with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS), to perform a novel comparison of expression profiles in a CMS pepper line

Cytoplasmic Male Sterility of Rice with Boro II Cytoplasm Is Caused by a Cytotoxic Peptide and Is Restored by Two Related PPR Motif Genes via Distinct Modes of mRNA Silencing[W

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Wang, Zhonghua; Zou, Yanjiao; Li, Xiaoyu; Zhang, Qunyu; Chen, Letian; Wu, Hao; Su, Dihua; Chen, Yuanling; Guo, Jingxin; Luo, Da; Long, Yunming; Zhong, Yang; Liu, Yao-Guang

2006-01-01

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic–nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function. PMID:16489123

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

International Nuclear Information System (INIS)

Espinosa, Alexander; Oke, Vilija; Elfving, Ase; Nyberg, Filippa; Covacu, Ruxandra; Wahren-Herlenius, Marie

2008-01-01

Patients with the systemic autoimmune diseases Sjoegrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus

The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

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Nikola Kellner

2014-01-01

Full Text Available The conserved NineTeen protein complex (NTC is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

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Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1975-12-01

The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

Cytoplasmic Copper Detoxification in Salmonella Can Contribute to SodC Metalation but Is Dispensable during Systemic Infection.

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Fenlon, Luke A; Slauch, James M

2017-12-15

Salmonella enterica serovar Typhimurium is a leading cause of foodborne disease worldwide. Severe infections result from the ability of S Typhimurium to survive within host immune cells, despite being exposed to various host antimicrobial factors. SodCI, a copper-zinc-cofactored superoxide dismutase, is required to defend against phagocytic superoxide. SodCII, an additional periplasmic superoxide dismutase, although produced during infection, does not function in the host. Previous studies suggested that CueP, a periplasmic copper binding protein, facilitates acquisition of copper by SodCII. CopA and GolT, both inner membrane ATPases that pump copper from the cytoplasm to the periplasm, are a source of copper for CueP. Using in vitro SOD assays, we found that SodCI can also utilize CueP to acquire copper. However, both SodCI and SodCII have a significant fraction of activity independent of CueP and cytoplasmic copper export. We utilized a series of mouse competition assays to address the in vivo role of CueP-mediated SodC activation. A copA golT cueP triple mutant was equally as competitive as the wild type, suggesting that sufficient SodCI is active to defend against phagocytic superoxide independent of CueP and cytoplasmic copper export. We also confirmed that a strain containing a modified SodCII, which is capable of complementing a sodCI deletion, was fully virulent in a copA golT cueP background competed against the wild type. These competitions also address the potential impact of cytoplasmic copper toxicity within the phagosome. Our data suggest that Salmonella does not encounter inhibitory concentrations of copper during systemic infection. IMPORTANCE Salmonella is a leading cause of gastrointestinal disease worldwide. In severe cases, Salmonella can cause life-threatening systemic infections, particularly in very young children, the elderly, or people who are immunocompromised. To cause disease, Salmonella must survive the hostile environment inside host

Origin of microbial life hypothesis: a gel cytoplasm lacking a bilayer membrane, with infrared radiation producing exclusion zone (EZ) water, hydrogen as an energy source and thermosynthesis for bioenergetics.

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Trevors, J T; Pollack, G H

2012-01-01

The hypothesis is proposed that pre-biotic bacterial cell(s) and the first cells capable of growth/division did not require a cytoplasmic membrane. A gel-like microscopic structure less than a cubic micrometer may have had a dual role as both an ancient pre-cytoplasm and a boundary layer to the higher-entropy external environment. The gel pre-cytoplasm exposed to radiant energy, especially in the infrared (IR) region of the EM spectrum resulted in the production of an exclusion zone (EZ) with a charge differential (-100 to -200 mV) and boundary that may have been a possible location for the latter organization of the first cytoplasmic membrane. Pre-biotic cells and then-living cells may have used hydrogen as the universal energy source, and thermosynthesis in their bioenergetic processes. These components will be discussed as to how they are interconnected, and their hypothesized roles in the origin of life. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Application of molecular markers in sugarcane germplasm innovation and breeding: new germplasm with cytoplasm from Saccharum spontaneum

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All current sugarcane cultivars (Saccharum hybrids spp.) are interspeci'c hybrids of S. of'cinarum, S. robustum and S. spontaneum that bear the same cytoplasm of S. of'cinarum. Until the end of 20th century, S. spontaneum was exclusively used as male parents to confer such traits as vigor, ratoonabi...

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

DEFF Research Database (Denmark)

Young, Samantha A M; Miyata, Haruhiko; Satouh, Yuhkoh

2016-01-01

manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression...

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

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Hatahet Feras

2010-09-01

Full Text Available Abstract Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3 pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

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Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

[Inheritance of reversions to male fertility in male-sterile sorghum hybrids with 9E cytoplasm male sterility induced by environmental conditions].

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Elkonin, L A; Gerashchenkov, G A; Domanina, I V; Rozhnova, N A

2015-03-01

Heritable phenotypic alterations occurring during plant ontogenesis under the influence of environmental factors are among the most intriguing genetic phenomena. It was found that male-sterile sorghum hybrids in the 9E cytoplasm from the F1 and F2 generations, which were obtained by crossing CMS lines with different fertile lines grown in field conditions, were transferred to greenhouse produce fertile tillers. Lines created by the self-pollination of revertant tillers exhibit complete male fertility upon cultivation under various environments (in the field, Tdry plot,(y) Tirrigated plot(y)). In a number of test-crosses of revertants to CMS lines in the 9E cytoplasm, restoration of male fertility in F1 hybrids was found, indicating that revertants possess functional fertility-restoring genes. A high positive correlation was found between the fertility level of the test-cross hybrids and the hydrothermal coefficient (the ratio of the sum of precipitation to the sum of temperatures) during the booting stage and pollen maturation (r = 0.75...0.91; Pmale fertility are due to up-regulation of fertility-restoring genes by a high level of water availability. Comparative MSAP-analysis of DNA of male-sterile and male-fertile test-cross hybrids using HpaII/MspI restrictases and primers to polygalacturonase gene ADPG2, which is required for cell separation during reproductive development, and gene MYB46, the transcription factor regulating secondary wall biosynthesis, revealed differences in the number and the length of amplified fragments. Changes in the methylation of these genes in conditions of drought stress are apparently the reason for male sterility of sorghum hybrids in the 9E cytoplasm. These data demonstrate that methylation of nuclear genes in sterility-inducing cytoplasm may be one of mechanisms causing the CMS phenomenon.

BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

Science.gov (United States)

The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

Functional analysis of the baculovirus 10 kilodalton protein

NARCIS (Netherlands)

Oers, van M.M.

1994-01-01

Nuclear polyhedrosis viruses belong to the family Baculoviridae and cause fatal diseases in arthropods predominantly in insects of the order Lepidoptera . These viruses were first reported in silk worms where viral infections could have devastating

Gas6 downregulation impaired cytoplasmic maturation and pronuclear formation independent to the MPF activity.

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Kyeoung-Hwa Kim

Full Text Available Previously, we found that the growth arrest-specific gene 6 (Gas6 is more highly expressed in germinal vesicle (GV oocytes than in metaphase II (MII oocytes using annealing control primer (ACP-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi. Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%. After stimulation with Sr(2+, Gas6-silenced MII oocytes had markedly reduced Ca(2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1 the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2 the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.

Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

International Nuclear Information System (INIS)

Ruel, Nancy; Zago, Anna; Spear, Patricia G.

2006-01-01

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

Science.gov (United States)

Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

2014-09-01

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis

International Nuclear Information System (INIS)

Savage, C.O.S.; Winearls, C.G.; Jones, S.; Marshall, P.D.; Lockwood, C.M.

1987-01-01

The diagnosis and management of Wegener's granulomatosis and microscopic polyarteritis are complicated by the lack of specific diagnostic tests. The diagnostic performance of a solid-phase radioimmunoassay, which detects the autoantibodies against neutrophil cytoplasm present in these disorders, was assessed in a prospective study of patients with suspected vasculitis and/or rapidly progressive nephritis. The assay had a sensitivity and specificity of 96% when carried out in combination with a specific inhibition stage and indirect immunofluorescence staining of alcohol-fixed normal neutrophils. (author)

Prediction of Chlamydia pneumoniae protein localization in host mitochondria and cytoplasm and possible involvements in lung cancer etiology: a computational approach

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Aws Alshamsan

2017-12-01

Full Text Available Collecting evidence suggests that the intercellular infection of Chlamydia pneumoniae in lungs contributes to the etiology of lung cancer. Many proteins of Chlamydia pneumoniae outmanoeuvre the various system of the host. The infection may regulate various factors, which can influence the growth of lung cancer in affected persons. In this in-silico study, we predict potential targeting of Chlamydia pneumoniae proteins in mitochondrial and cytoplasmic comportments of host cell and their possible involvement in growth and development of lung cancer. Various cellular activities are controlled in mitochondria and cytoplasm, where the localization of Chlamydia pneumoniae proteins may alter the normal functioning of host cells. The rationale of this study is to find out and explain the connection between Chlamydia pneumoniae infection and lung cancer. A sum of 183 and 513 proteins were predicted to target in mitochondria and cytoplasm of host cell out of total 1112 proteins of Chlamydia pneumoniae. In particular, many targeted proteins may interfere with normal growth behaviour of host cells, thereby altering the decision of program cell death. Present article provides a potential connection of Chlamydia pneumoniae protein targeting and proposed that various targeted proteins may play crucial role in lung cancer etiology through diverse mechanisms.

Agronomic Performance of Flue-Cured Tobacco F1 Hybrids Obtained with Different Sources of Male Sterile Cytoplasm

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Berbec A

2014-12-01

Full Text Available Four cytoplasmic male sterile (cms F1flue-cured hybrids cv. Wiaelica × cv. Virginia Golta (VG, the male fertile analogue and the parental varieties were tested at two locations in Poland in a replicated field trial. The cms sources in the hybrids wereN. suaveolens,N. amplexicaulis,N. bigeloviiand aN. tabacumcms mutant. Under the slight to moderate pressure from black root rot present at the trial sites the hybrids showed a moderate tolerance of the disease characteristic of VG as opposed to medium strong susceptibility of Wislica. Apart from the effect of black root rot tolerance the vegetative vigor of the hybrids (plant height, leaf size, earliness was affected by cytoplasm source. The F1hybrid withN. suaveolens cytoplasm flowered approximately three days later than the remaining hybrids. Of the cms hybrids tested cmsN. bigelovii produced the tallest plants with largest mid-position leaves. Yields of cured leaves were largely influenced by black root rot and were generally higher in VG and in the hybrids than in Wislica. Leaf yields and curability were generally little affected by cms source under low pressure from black root rot. At the site with a relatively high level of black root rot infestation the yields of cmsN. suaveolens were slightly lower but the percentage of light grades slightly higher compared to those of other cms hybrids. CmsN. suaveolens was the best hybrid in terms of money returns at the low black root rot field but it was the poorest hybrid performer under high pressure from the disease. Contents of nitrogen, sugars, nicotine and ash was little affected by source of cms. There was an increased incidence of potato virus Y (PVY and white spots in cmsN. suaveolens and, to a lesser extent, in cmsN. bigelovii as compared to the remaining disease-free entries.

Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

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Ashley T Martino

2009-08-01

Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

Science.gov (United States)

Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

2012-09-18

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

Antineutrophil cytoplasm antibody: positivity and clinical correlation.

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Martínez Téllez, Goitybell; Torres Rives, Bárbara; Rangel Velázquez, Suchiquil; Sánchez Rodríguez, Vicky; Ramos Ríos, María Antonia; Fuentes Smith, Lisset Evelyn

2015-01-01

To determine positivity and clinical correlation of anti-neutrophil cytoplasmic antibodies (ANCA), taking into account the interference of antinuclear antibodies (ANA). A prospective study was conducted in the Laboratory of Immunology of the National Cuban Center of Medical Genetic during one year. Two hounded sixty-seven patients with indication for ANCA determination were included. ANCA and ANA determinations with different cut off points and assays were determined by indirect immunofluorescense. Anti proteinase 3 and antimyeloperoxidase antibodies were determined by ELISA. Most positivity for ANCA was seen in patients with ANCA associated, primary small-vessel vasculitides, rheumatoid arthritis and systemic lupus erythematosus. Presence of ANCA without positivity for proteinase 3 and myeloperoxidase was higher in patients with ANA and little relation was observed between the perinuclear pattern confirmed in formalin and specificity by myeloperoxidase. Highest sensibility and specificity values for vasculitides diagnostic were achieved by ANCA determination using indirect immunofluorescense with a cut off 1/80 and confirming antigenic specificities with ELISA. ANCA can be present in a great number of chronic inflammatory or autoimmune disorders in the population studied. This determination using indirect immunofluorescence and following by ELISA had a great value for vasculitis diagnosis. Anti mieloperoxidasa assay has a higher utility than the formalin assay when ANA is present. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

Constitutively expressed Protocadherin-α regulates the coalescence and elimination of homotypic olfactory axons through its cytoplasmic region

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Sonoko eHasegawa

2012-10-01

Full Text Available Olfactory sensory neuron (OSN axons coalesce into specific glomeruli in the olfactory bulb (OB according to their odorant receptor (OR expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α’s cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

International Nuclear Information System (INIS)

Heude, M.; Chanet, R.

1975-01-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid-held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin and chloramphenicol. It was shown that mitochondrial proteins are involved in the recovery and survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the e + genotype in UV-irradiated dark liquid-held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid-holding process for the e - induction, as shown by inhibiting mitochondrial protein synthesis of both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid-holding of the UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage in particular is not correlated with the repressed or derepressed state of the mitochondria

Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

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Enrique Álvarez

Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

Anti-neutrophil cytoplasmic antibody-associated vasculitis with renal involvement: Analysis of 89 cases.

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Caravaca-Fontán, Fernando; Yerovi, Estefanía; Delgado-Yagu E, María; Galeano, Cristina; Pampa-Saico, Saúl; Tenorio, Maria Teresa; Liaño, Fernando

2017-01-06

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with renal involvement are associated with high morbi-mortality. In this study we analyse if the prognosis of these diseases have improved in recent years, and which factors influence the outcomes. Retrospective single-centre observational study, which included all patients diagnosed with microscopic polyangiitis and granulomatosis with polyangiitis with renal involvement in the last 25 years. Demographic, clinical and biochemical parameters of prognostic interest were recorded. The differences between four chronological periods were analysed, along with the determinants of a poor outcome (death or end-stage renal disease). Eighty-nine patients were included (mean age 64±15 years). Sixty-four patients (72%) had microscopic polyangiitis and 25 (28%) granulomatosis with polyangiitis. During the study period, 37 (42%) patients died. Through Cox regression analysis, the best determinants of mortality were the initial glomerular filtration rate (HR 0.911; P=.003), Charlson comorbidity index (HR 1.513; P<.0001) and tobacco smoking (HR 1.816; P=.003). 35% developed end-stage renal disease, and the best determinants (by competing-risk regression) were: initial glomerular filtration rate (sub-hazard ratio [SHR]: 0.791; P<.0001), proteinuria (SHR: 1.313; P<.0001), and smoking status (SHR: 1.848; P=.023). No differences were found in patients' mortality or renal survival between the different study periods. Prognosis of anti-neutrophil cytoplasm antibodies vasculitis with renal involvement treated with conventional immunosuppressive therapy remains unsatisfactory, and continues to have increased long-term complications and mortality. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

International Nuclear Information System (INIS)

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

2008-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

Tapetal-Delayed Programmed Cell Death (PCD and Oxidative Stress-Induced Male Sterility of Aegilops uniaristata Cytoplasm in Wheat

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Zihan Liu

2018-06-01

Full Text Available Cytoplasmic male sterility (CMS plays a crucial role in the utilization of hybrid vigor. Pollen development is often accompanied by oxidative metabolism responses and tapetal programmed cell death (PCD, and deficiency in these processes could lead to male sterility. Aegilops uniaristata cytoplasmic male sterility (Mu-CMS wheat is a novel male-sterile line in wheat, which possess important potential in hybrid wheat breeding. However, its CMS mechanisms remain poorly understood. In our study, U87B1-706A, with the Aegilops uniaristata cytoplasm, and the maintainer line 706B were used to explore the abortive reason. Compared with 706B, histological analysis and PCD detection of the anther demonstrated that U87B1-706A appeared as delayed tapetal PCD as well as a disorganized organelle phenotype in the early uninucleate stage. Subsequently, a shrunken microspore and disordered exine structure were exhibited in the late uninucleate stage. While the activities of antioxidase increased markedly, the nonenzymatic antioxidant contents declined obviously following overacummulation of reactive oxygen species (ROS during pollen development in U87B1-706A. Real-time quantitative PCR testified that the transcript levels of the superoxide dismutase (SOD, catalase (CAT, and ascorbate peroxidase (APX genes, encoding pivotal antioxidant enzymes, were up-regulated in early pollen development. Therefore, we deduce excess ROS as a signal may be related to the increased expression levels of enzyme genes, thereby breaking the antioxidative system balance, resulting in delayed tapetal PCD initiation, which finally led to pollen abortion and male sterility in U87B1-706A. These results provide evidence to further explore the mechanisms of abortive pollen in CMS wheat.

Implications of Antiphospholipid and Antineutrophilic Cytoplasmic Antibodies in the Context of Postinfectious Glomerulonephritis

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Daniel Leifer

2017-01-01

Full Text Available While antineutrophil cytoplasmic antibody (ANCA positivity has been documented in some patients with postinfectious glomerulonephritis (PIGN and is associated with more severe disease, antiphospholipid antibodies (APA are not known to be a common occurrence. We describe a child with severe acute kidney injury who was noted to have prolonged positivity of both ANCA and APA; a renal biopsy showed noncrescentic immune complex mediated glomerulonephritis with subepithelial deposits compatible with PIGN. He recovered without maintenance immunosuppressive therapy and at last follow-up had normal renal function. We discuss the cooccurrence and implications of ANCA and APA in children with PIGN.

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

International Nuclear Information System (INIS)

Shimura, Takaya; Higashiyama, Shigeki; Joh, Takashi; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi

2012-01-01

Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

Science.gov (United States)

2012-01-01

Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Conclusions Both the function of HB-EGF as an EGFR ligand

Effect of physical and chemical mutagens and male sterile cytoplasm of chaisma frequency in pearl millet inbreds and hybrids

International Nuclear Information System (INIS)

Singh, B.D.; Singh, R.B.; Singh, R.M.; Vijay Laxmi

1977-01-01

Chiasma frequency was recorded in normal and treated [10, 20, 30 Kr γ-rays, 0.2% ethyl methane-sulphonate (EMS) and 10 Kr γ-rays + 0.2% EMS] populations of 7 inbreds and 3 hybrids of pearl millet. Inbreds in general showed lower chiasma frequency than hybrids. However, inbred Bi13B showed the highest chiasma frequency. The male sterile cytoplasm reduced the chaisma frequency and increased the among-plant-variability in the inbreds and, therefore, possibly in the hybrids which had male sterile cytoplasm. γ-rays were more effective than EMS in reducing chiasma frequency. In most of the genotypes 10Kr γ-rays and 0.2% EMS promoted chiasma frequency. The combination treatments showed greater effect than γ-rays and EMS applied individually. Hybrids as a group, showed lower variation for chiasma number than inbreds in response to the mutagenic treatments. (author)

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus

Energy Technology Data Exchange (ETDEWEB)

Soonthornsit, Jeerawat [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Yamaguchi, Yoko; Tamura, Daisuke [Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Yamamoto, Akitsugu [Department of Animal Bioscience, Nagahama Institute of Bio-Science and Technology, 266 Tamura, Nagahama, Shiga, 526‐0829 (Japan); Nakamura, Nobuhiro, E-mail: osaru3@cc.kyoto-su.ac.jp [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)

2014-11-01

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1–2 h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A{sub 2} inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A{sub 2} was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. - Highlights: • The Golgi apparatus reversibly disassembles by low pH treatment. • The cis-Golgi disassembles quickly generating tubular structures. • Both anterograde and retrograde transport between the ER and the Golgi apparatus are reduced. • Phospholipase A{sub 2} inhibitors (ONO

Rapid and Sustained Nuclear-Cytoplasmic ERK Oscillations Induced by Epidermal Growth Factor

Energy Technology Data Exchange (ETDEWEB)

Shankaran, Harish; Ippolito, Danielle L.; Chrisler, William B.; Resat, Haluk; Bollinger, Nikki; Opresko, Lee K.; Wiley, H. S.

2009-12-01

Mathematical modeling has predicted that ERK activity should oscillate in response to cell stimulation, but this has never been observed. To explore this inconsistency, we expressed an ERK1-GFP fusion protein in mammary epithelial cells. Following EGF stimulation, we observed rapid and continuous ERK oscillations between the nucleus and cytoplasm with a periodicity of approximately 15 minutes. These oscillations were remarkably persistent (>45 cycles), displayed an asymmetric waveform, and were highly dependent on cell density, essentially disappearing at confluency. We conclude that the ERK pathway is an intrinsic oscillator. Although the functional implications of the observed oscillations are uncertain, this property can be used to continuously monitor ERK activity in single cells.

Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

LENUS (Irish Health Repository)

Hong, Ying

2012-01-01

The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

Science.gov (United States)

Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

2016-07-01

Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Inhibitiory properties of cytoplasmic extract of Lactobacilli isolated from common carp intestine on human chronic myelocytic leukemia K562 cell line: an in vitro study

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Kabiri F

2011-03-01

Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Lactobacillus species are genetically diverse groups of Lactic Acid Bacteria (LAB that have been introduced as probiotics, because of some characteristics such as their anti-tumor properties, helping the intestinal flora balance, production of antibiotics, stimulation of host immune response, etc. The aim of this study was to investigate the effects of cytoplasmic extraction and cell wall of Lactobacillus species isolated from the intestine of common carp on human chronic myelocytic leukemia or K562 cancer cell lines."n"nMethods: The intestinal contents of 115 common carp captured from the natural resources of West Azerbaijan province in Iran were examined for LAB. After isolation, the identification of Lactobacilli was done according to traditional and molecular bacteriological tests. Subsequently, a suspension of each bacterium was prepared and the protein content of the cytoplasm was extracted. Cell wall disintegration was done by cell lysis buffer and sonication. The effects of cytoplasmic extraction and cell wall on K562 cell line proliferation were investigated by MTT assays."n"nResults: The cytoplasmic extraction of the isolated Lactobacilli had significant (p<0.05 anti

Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

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Aleksandra Glogowska

2012-05-01

Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

Disease-parasitoid relationships in natural populations of Lymantria dispar [Lep.: Lymantriidae] in the northeastern United States

Science.gov (United States)

R.C. Reardon; J.D. Podgwaite

1976-01-01

Immature Lymantria dispar L. were collected from 6 geographically distinct populations over 2 years to determine correlations between parasitoid and disease incidences. Incidence of the nuclear polyhedrosis virus disease (NPV) was found to be positively correlated with incidences of the parasitoids Apanteles melanoscelus (Ratzeburg...

Characterization of Baculovirus Insecticides Expressing Tailored Bacillus thuringiensis CryIA(b) Crystal Proteins

NARCIS (Netherlands)

Martens, John W M; Knoester, Marga; Weijts, Franci; Groffen, Sander J A; Hu, Zhihong; Bosch, Dirk; Vlak, Just M.

1995-01-01

Full-length, truncated, and mature forms of the CryIA(b) insecticidal crystal protein gene of Bacillus thuringiensis were engineered into the p10 locus of Autographa californica nuclear polyhedrosis virus (AcNPV). A signal sequence of Heliothis virescens juvenile hormone esterase was introduced at

Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

DEFF Research Database (Denmark)

Rosorius, O; Issinger, O G; Braulke, T

1993-01-01

The cytoplasmic tail of the human 300-kDa mannose 6-phosphate receptor (MPR 300-CT) is an excellent substrate for casein kinase II in vitro. The phosphorylated MPR 300-CT was cross-linked by means of bis(sulfosuccinimidyl)suberate mainly to a cytosolic protein of 35 kDa (referred to as TIP 35...... with TIP 35 is phosphorylation-specific. Furthermore, TIP 35 was only cross-linked to the MPR 300-CT phosphorylated by casein kinase II whereas the MPR 300-CT phosphorylated by protein kinase A failed to cross-link to TIP 35. These results indicate that the cytoplasmic tail of the MPR 300 interacts...

Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

Science.gov (United States)

Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

2017-12-13

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.

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Mezzalira, Joana Claudia; Ohlweiler, Lain Uriel; da Costa Gerger, Renato Pereira; Casali, Renata; Vieira, Fabiano Koerich; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Rodrigues, José Luiz; Mezzalira, Alceu; Bertolini, Marcelo

2011-02-01

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Sexy sons: a dead end for cytoplasmic genes.

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Zeh, Jeanne A

2004-08-07

Critics of sexual conflict theory argue that females may gain a net reproductive benefit from mating with manipulative males because the direct costs that they suffer may be offset by the production of sexy, i.e. manipulative, sons. However, this exclusive focus on nuclear gene effects represents an incomplete view of female fitness. Females differ fundamentally from males in transmitting not only nuclear genes but also a wide range of cytoplasmic genetic elements (CGEs) that can have profound effects, from male killing to influencing development of the nervous system and cognitive ability. Maternal transmission of CGEs has two major implications for sexual selection. First, the evolution of male fitness traits, such as sperm competitive ability, may be constrained because response to selection on mitochondrial genomes can occur only through the female line. Second, CGEs bear the direct costs of male manipulation but gain no indirect benefits when females produce sexy sons. This should result in perpetual antagonistic coevolution between nuclear genes involved in male manipulation and CGEs that promote female resistance to male sexually selected traits. Explicit consideration of the consequences of selection acting on CGEs is therefore necessary for a better understanding of the relationship between sexual selection and sexual conflict.

Changes in Cellular mRNA Stability, Splicing, and Polyadenylation through HuR Protein Sequestration by a Cytoplasmic RNA Virus

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Michael D. Barnhart

2013-11-01

Full Text Available The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3′ UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3′ UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

A case of propylthiouracil-induced antineutrophilic cytoplasmic antibody-positive vasculitis successfully treated with radioactive iodine

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C. Bes

2013-07-01

Full Text Available Antineutrophilic cytoplasmic antibody (ANCA associated vasculitis is one of the rare complications of propylthiouracil treatment. Having a variable clinical spectrum, it may be presented with both skin limited vasculitis and life-threatening systemic vasculitis. In this study, we present a case that developed ANCA-positive vasculitis with skin and kidney involvement (hematuria and proteinuria six months after propylthiouracil treatment was initiated for toxic nodular goiter. Proteinuria recovered dramatically subsequent to radioactive iodine treatment following ceasing the drug.

Development of Cytoplasmic Male Sterile IR24 and IR64 Using CW-CMS/Rf17 System.

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Toriyama, Kinya; Kazama, Tomohiko

2016-12-01

A wild-abortive-type (WA) cytoplasmic male sterility (CMS) has been almost exclusively used for breeding three-line hybrid rice. Many indica cultivars are known to carry restorer genes for WA-CMS lines and cannot be used as maintainer lines. Especially elite indica cultivars IR24 and IR64 are known to be restorer lines for WA-CMS lines, and are used as male parents for hybrid seed production. If we develop CMS IR24 and CMS IR64, the combination of F1 pairs in hybrid rice breeding programs will be greatly broadened. For production of CMS lines and restorer lines of IR24 and IR64, we employed Chinese wild rice (CW)-type CMS/Restorer of fertility 17 (Rf17) system, in which fertility is restored by a single nuclear gene, Rf17. Successive backcrossing and marker-assisted selection of Rf17 succeeded to produce completely male sterile CMS lines and fully restored restorer lines of IR24 and IR64. CW-cytoplasm did not affect agronomic characteristics. Since IR64 is one of the most popular mega-varieties and used for breeding of many modern varieties, the CW-CMS line of IR64 will be useful for hybrid rice breeding.

The Structure of the ZMYND8/Drebrin Complex Suggests a Cytoplasmic Sequestering Mechanism of ZMYND8 by Drebrin.

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Yao, Ningning; Li, Jianchao; Liu, Haiyang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2017-11-07

Malfunctions of the actin binding protein Drebrin have been implicated in various human diseases such as Alzheimer's disease, cognitive impairments, cancer, and digestive disorders, though with poorly understood mechanisms. The ADF-H domain of Drebrin does not contain actin binding and depolymerizing activity. Instead, it binds to a histone marker reader, ZMYND8. Here we present the high-resolution crystal structure of Drebrin ADF-H in complex with the ZMYND8 PHD-BROMO-PWWP tandem, elucidating the mechanistic basis governing the highly specific interaction of the two proteins. The structure reveals that the ZMYND8 PHD-BROMO-PWWP tandem forms a structural supramodule that is necessary for binding to Drebrin ADF-H. Drebrin ADF-H competes with modified histone for binding to ZMYND8. Binding of Drebrin can shuttle ZMYND8 from nucleus to cytoplasm in living cells. Taken together, our study uncovers a non-actin target binding mode for ADF-H domains, and suggests that Drebrin may regulate activities of epigenetic reader ZMYND8 via its cytoplasmic sequestration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stage-specific appearance of cytoplasmic microtubules around the surviving nuclei during the third prezygotic division of Paramecium.

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Wang, Yi-Wen; Yuan, Jin-Qiang; Gao, Xin; Yang, Xian-Yu

2012-12-01

There are six micronuclear divisions during conjugation of Paramecium caudatum: three prezygotic and three postzygotic divisions. Four haploid nuclei are formed during the first two meiotic prezygotic divisions. Usually only one meiotic product is located in the paroral cone (PC) region at the completion of meiosis, which survives and divides mitotically to complete the third prezygotic division to yield a stationary and a migratory pronucleus. The remaining three located outside of the PC degenerate. The migratory pronuclei are then exchanged between two conjugants and fuse with the stationary pronuclei to form synkarya, which undergo three successive divisions (postzygotic divisions). However, little is known about the surviving mechanism of the PC nuclei. In the current study, stage-specific appearance of cytoplasmic microtubules (cMTs) was indicated during the third prezygotic division by immunofluorescence labeling with anti-alpha tubulin antibodies surrounding the surviving nuclei, including the PC nuclei and the two types of prospective pronuclei. This suggested that cMTs were involved in the formation of a physical barrier, whose function may relate to sequestering and protecting the surviving nuclei from the major cytoplasm, where degeneration of extra-meiotic products occurs, another important nuclear event during the third prezygotic division.

Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana BRI1-associated kinase 1 (BAK1) cytoplasmic domain

International Nuclear Information System (INIS)

Gao, Jian; Ma, Yuanyuan; Sun, Yuna; Zhao, Huadong; Hong, Dapeng; Yan, Liming; Lou, Zhiyong

2012-01-01

The cytoplasmic domain of BRI1-associated kinase 1 from A. thaliana has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution. BRI1-associated kinase 1 (BAK1) is a member of the plant receptor-like kinase (RLK) superfamily. BAK1 has been shown to initiate brassinosteroid (BR) signalling and innate immune responses in plants by forming receptor complexes with both brassinosteroid-insensitive 1 (BRI1) and flagellin-sensing 2 (FLS2). To gain a better understanding of the structural details and the mechanism of action of the BAK1 kinase domain, recombinant BAK1 cytoplasmic domain has been expressed, purified and crystallized at 291 K using PEG 3350 as a precipitant. A 2.6 Å resolution data set was collected from a single flash-cooled crystal at 100 K. This crystal belonged to space group C2, with unit-cell parameters a = 70.3, b = 75.6, c = 71.9 Å, β = 93.1°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient was 2.6 Å 3 Da −1

iTRAQ-Based Proteomics Analyses of Sterile/Fertile Anthers from a Thermo-Sensitive Cytoplasmic Male-Sterile Wheat with Aegilops kotschyi Cytoplasm

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Gaoming Zhang

2018-05-01

Full Text Available A “two-line hybrid system” was developed, previously based on thermo-sensitive cytoplasmic male sterility in Aegilops kotschyi (K-TCMS, which can be used in wheat breeding. The K-TCMS line exhibits complete male sterility and it can be used to produce hybrid wheat seeds during the normal wheat-growing season; it propagates via self-pollination at high temperatures. Isobaric tags for relative and absolute quantification-based quantitative proteome and bioinformatics analyses of the TCMS line KTM3315A were conducted under different fertility conditions to understand the mechanisms of fertility conversion in the pollen development stages. In total, 4639 proteins were identified, the differentially abundant proteins that increased/decreased in plants with differences in fertility were mainly involved with energy metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, protein synthesis, translation, folding, and degradation. Compared with the sterile condition, many of the proteins that related to energy and phenylpropanoid metabolism increased during the anther development stage. Thus, we suggest that energy and phenylpropanoid metabolism pathways are important for fertility conversion in K-TCMS wheat. These findings provide valuable insights into the proteins involved with anther and pollen development, thereby, helping to further understand the mechanism of TCMS in wheat.

Ultraviolet mutagenesis studies of [psi], a cytoplasmic determinant of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Tuite, M.F.; Cox, B.S.

1980-01-01

uv mutagenesis was used to probe the molecular nature of [psi], a nonmitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The uv-induced mutation from [psi + ] to [psi - ] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi - ] mutation was demonstrated by photoreactivation. uv-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. uv-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP).

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Baratella, Marco; Forlani, Greta; Raval, Goutham U; Tedeschi, Alessandra; Gout, Olivier; Gessain, Antoine; Tosi, Giovanna; Accolla, Roberto S

2017-01-01

HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP.

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Marco Baratella

2017-01-01

Full Text Available HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL, and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP. Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Acute respiratory failure as primary manifestation of antineutrophil cytoplasmic antibodies-associated vasculitis

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Evdokia Sourla

2014-07-01

Full Text Available The systemic vasculitides are multifocal diseases characterized by the presence of blood vessel inflammation in multiple organ systems. Their clinical presentation is variable extending from self-limited illness to critical complications including diffuse alveolar hemorrhage and glomerulonephritis. Alveolar hemorrhage is a lifethreatening manifestation of pulmonary vasculitis that can rapidly progress into acute respiratory failure requiring ventilatory support. We present the case of a 74-year-old patient admitted to the Intensive Care Unit with severe hypoxic respiratory failure and diffuse alveolar infiltrates in chest imaging that was later diagnosed as antineutrophil cytoplasmic antibodies-associated vasculitis. The report highlights the importance of differentiate between alveolar hemorrhage and acute respiratory distress syndrome of other etiology because alveolar hemorrhage is reversible with prompt initiation of treatment.

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

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Xu, Pei; Yang, Yuwen; Zhang, Zhengzhi; Chen, Weihua; Zhang, Caiqin; Zhang, Lixia; Zou, Sixiang; Ma, Zhengqiang

2008-01-01

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

International Nuclear Information System (INIS)

Saha, Kunal; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

2005-01-01

Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis

Microtubule plus end-tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans.

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Zeng, Cui J Tracy; Kim, Hye-Ryun; Vargas Arispuro, Irasema; Kim, Jung-Mi; Huang, An-Chi; Liu, Bo

2014-11-01

Cytoplasmic microtubules (MTs) serve as a rate-limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end-tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end-tracking of CLIPA, the Kinesin-7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi. © 2014 John Wiley & Sons Ltd.

Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

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Imam, Zachary I; Kenyon, Laura E; Ashby, Grant; Nagib, Fatema; Mendicino, Morgan; Zhao, Chi; Gadok, Avinash K; Stachowiak, Jeanne C

2017-10-01

From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes. Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and

Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.

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Gullmets, Josef; Torvaldson, Elin; Lindqvist, Julia; Imanishi, Susumu Y; Taimen, Pekka; Meinander, Annika; Eriksson, John E

2017-12-01

Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila , transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila , a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila , we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin. © FASEB.

Antineutrophil Cytoplasmic Antibodies Associated With Infective Endocarditis

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Langlois, Vincent; Lesourd, Anais; Girszyn, Nicolas; Ménard, Jean-Francois; Levesque, Hervé; Caron, Francois; Marie, Isabelle

2016-01-01

Abstract To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA. Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed. Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis (P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients. Our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. PMID:26817911

CanVasc recommendations for the management of antineutrophil cytoplasm antibody (ANCA)-associated vasculitides - Executive summary.

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McGeoch, Lucy; Twilt, Marinka; Famorca, Leilani; Bakowsky, Volodko; Barra, Lillian; Benseler, Susan; Cabral, David A; Carette, Simon; Cox, Gerald P; Dhindsa, Navjot; Dipchand, Christine; Fifi-Mah, Aurore; Goulet, Michele; Khalidi, Nader; Khraishi, Majed M; Liang, Patrick; Milman, Nataliya; Pineau, Christian A; Reich, Heather; Samadi, Nooshin; Shojania, Kam; Taylor-Gjevre, Regina; Towheed, Tanveer E; Trudeau, Judith; Walsh, Michael; Yacyshyn, Elaine; Pagnoux, Christian

2015-01-01

The Canadian Vasculitis research network (CanVasc) is composed of physicians from different medical specialties, including rheumatology and nephrology and researchers with expertise in vasculitis. One of its aims was to develop recommendations for the diagnosis and management of antineutrophil cytoplasm antibody (ANCA)-associated vasculitides in Canada. This executive summary features the 19 recommendations and 17 statements addressing general AAV diagnosis and management, developed by CanVasc group based on a synthesis of existing international guidelines, other published supporting evidence and expert consensus considering the Canadian healthcare context.

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.

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Wendy C Carcamo

Full Text Available Cytoplasmic filamentous rods and rings (RR structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.Distinct cytoplasmic rods (∼3-10 µm in length and rings (∼2-5 µm in diameter in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1 and inosine monophosphate dehydrogenase 2 (IMPDH2 were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%; upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

Members of the HCMV US12 family of predicted heptaspanning membrane proteins have unique intracellular distributions, including association with the cytoplasmic virion assembly complex

International Nuclear Information System (INIS)

Das, Subhendu; Pellett, Philip E.

2007-01-01

The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress

Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA

DEFF Research Database (Denmark)

Lubas, Michal Szymon; Damgaard, Christian Kroun; Tomecki, Rafal

2013-01-01

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes...

Cytoplasmic peptide:N-glycanase cleaves N-glycans on a carboxypeptidase Y mutant during ERAD in Saccharomyces cerevisiae.

Science.gov (United States)

Hosomi, Akira; Suzuki, Tadashi

2015-04-01

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected. We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity. The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404. The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo. This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged. Copyright © 2014 Elsevier B.V. All rights reserved.

Occurrence of antineutrophil cytoplasmic antibodies and associated vasculitis in patients with hyperthyroidism treated with antithyroid drugs : A long-term followup study

NARCIS (Netherlands)

Slot, MC; Links, TP; Stegeman, CA; Tervaert, JWC

2005-01-01

Objective. To test whether antineutrophil cytoplasmic antibodies (ANCA) and ANCA-associated vasculitis (AAV) are not only induced during treatment with antithyroid drugs, but can also become evident when medication has been ceased, possibly after years. Methods. Patients who visited our hospital for

Different reparability of the chromosomal and cytoplasmic deoxyribonucleic acid in Escherichia coli damaged by γ and ultraviolet irradiation

International Nuclear Information System (INIS)

Petranovic, D.; Petranovic, M.; Nozinic, R.; Trgovcevic, Z.

1978-01-01

The relative efficiencies by which chromosomal and extrachromosomal DNAs are repaired in irradiated bacteria were assayed. Repair-proficient Escherichia coli C600 cells lysogenic for, or infected with, the thermoinducible phage lambdacI857 ind were exposed to γ or uv radiation and then tested for colony- and plaque-forming ability. The results show that the bacterial cell is about 5 times more sensitive to γ rays and about 1.5 times more sensitive to uv light, if compared to either (1) the prophage that is irradiated in the bacterial chromosome and, on heat induction, repaired in the cytoplasm or (2) the infecting phage that is irradiated and repaired in the cytoplasm. Since the bacterial DNA is about 80 times larger than the phage DNA, it is inferred that repair processes operating along the chromosomal DNA are one order of magnitude more efficient than those operating along the extrachromosomal DNA. This conclusion is reinforced by the fact that the absence of repair in the system Escherichia coli AB2480 uvrA recA-lambdacI857 ind red gives the expected ratio of 80/1 for the uv sensitivity of cells and that of intracellular phage

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

Science.gov (United States)

Nicolas, William J; Grison, Magali S; Trépout, Sylvain; Gaston, Amélia; Fouché, Mathieu; Cordelières, Fabrice P; Oparka, Karl; Tilsner, Jens; Brocard, Lysiane; Bayer, Emmanuelle M

2017-06-12

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

Science.gov (United States)

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

Genetically-engineered baculovirus pesticides and their environmental safety

Science.gov (United States)

H. Alan Wood; Yu Zailin

1991-01-01

Baculoviruses such as the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) are ecologically attractive alternatives to chemical insect pesticides but have a slow rate of control. To overcome this we have developed and are field testing an environmentally acceptable strategy which can be used for the introduction and expression of pesticide-...

The Cytoplasmic Carbonic Anhydrases βCA2 and βCA4 Are Required for Optimal Plant Growth at Low CO2.

Science.gov (United States)

DiMario, Robert J; Quebedeaux, Jennifer C; Longstreth, David J; Dassanayake, Maheshi; Hartman, Monica M; Moroney, James V

2016-05-01

Carbonic anhydrases (CAs) are zinc metalloenzymes that interconvert CO2 and HCO3 (-) In plants, both α- and β-type CAs are present. We hypothesize that cytoplasmic βCAs are required to modulate inorganic carbon forms needed in leaf cells for carbon-requiring reactions such as photosynthesis and amino acid biosynthesis. In this report, we present evidence that βCA2 and βCA4 are the two most abundant cytoplasmic CAs in Arabidopsis (Arabidopsis thaliana) leaves. Previously, βCA4 was reported to be localized to the plasma membrane, but here, we show that two forms of βCA4 are expressed in a tissue-specific manner and that the two proteins encoded by βCA4 localize to two different regions of the cell. Comparing transfer DNA knockout lines with wild-type plants, there was no reduction in the growth rates of the single mutants, βca2 and βca4 However, the growth rate of the double mutant, βca2βca4, was reduced significantly when grown at 200 μL L(-1) CO2 The reduction in growth of the double mutant was not linked to a reduction in photosynthetic rate. The amino acid content of leaves from the double mutant showed marked reduction in aspartate when compared with the wild type and the single mutants. This suggests the cytoplasmic CAs play an important but not previously appreciated role in amino acid biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Genetics and biology of cytoplasmic male sterility (CMS) and its applications in forage and turf grass breeding

OpenAIRE

Islam Mohamed Shofiqul; Studer Bruno; Møller Ian Max; Asp Torben

2014-01-01

Hybrid breeding can exploit heterosis and thus offers opportunities to maximize yield quality and resistance traits in crop species. Cytoplasmic male sterility (CMS) is a widely applied tool for efficient hybrid seed production. Encoded in the mitochondrial genome CMS is maternally inherited and thus it can be challenging to apply in breeding schemes of allogamous self incompatible plant species such as perennial ryegrass. Starting with a general introduction to the origin and the function of...

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

KAUST Repository

Park, Hyo-Young

2017-04-21

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3\\' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins\\' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

KAUST Repository

Park, Hyo-Young; Lee, Keh Chien; Jang, Yun Hee; Kim, SoonKap; Thu, May Phyo; Lee, Jeong Hwan; Kim, Jeong-Kook

2017-01-01

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

Science.gov (United States)

Dorjgotov, Dulguun; Jurca, Manuela E; Fodor-Dunai, Csilla; Szucs, Attila; Otvös, Krisztina; Klement, Eva; Bíró, Judit; Fehér, Attila

2009-04-02

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

International Nuclear Information System (INIS)

Kutty, R. Krishnan; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

2006-01-01

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 27 KKRKAP 276 ) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting

Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

Science.gov (United States)

Grayson, Peter C.; Sloan, J. Mark; Niles, John L.; Monach, Paul A.; Merkel, Peter A.

2011-01-01

Objectives Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Methods Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 – October 2010. A PubMed database search of the medical literature was performed for papers published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. Results A 49 year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a post-recovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. Conclusions ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV. PMID:21507463

A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

Directory of Open Access Journals (Sweden)

Ashley L Waldron

Full Text Available Histidyl tRNA Synthetase (HARS is a member of the aminoacyl tRNA synthetase (ARS family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

Directory of Open Access Journals (Sweden)

Tentler John J

2011-08-01

Full Text Available Abstract Background The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. Results To map the minimal functional nuclear localization (NLS and nuclear export (NES signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247 in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112 and another to the DNA binding domain (DBD, (NES2: 275LWEFIRDILI284. Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323 by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. Conclusions These data highlight that ESE-1 contains NLS and NES signals that play a critical role in

Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

Science.gov (United States)

Mizukami, Maki; Yamada, Misaki; Fukui, Sayaka; Fujimoto, Nao; Yoshida, Shigeru; Kaga, Sanae; Obata, Keiko; Jin, Shigeki; Miwa, Keiko; Masauzi, Nobuo

2016-11-01

Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image-J (IJ), called PS-IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS-IJ. Whole-blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color-tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS-IJ, the rAV/rECA ratios calculated with results from PS-IJ were compared with those calculated with human eye and IJ (HE-IJ). The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS-IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE-IJ. PS-IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film. © 2016 Wiley Periodicals, Inc.

Genetic Analysis of Embryo, Cytoplasm and Maternal Effects and Their Environment Interactions for Isoflavone Content in Soybean [Glycine max(L.) Merr.

Institute of Scientific and Technical Information of China (English)

无

2007-01-01

Soybean seed products contain isoflavones (genistein, daidzein, and glycitein) that display biological effects when ingested by humans and animals. These effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key factor to the biological effect. Our objective was to identify the genetic effects that underlie the isoflavone content in soybean seeds. A genetic model for quantitative traits of seeds in diploid plants was applied to estimate the genetic main effects and genotype × environment (GE) interaction effects for the isoflavone content (IC) of soybean seeds by using two years experimental data with an incomplete diallel mating design of six parents. Results showed that the IC of soybean seeds was simultaneously controlled by the genetic effects of maternal,embryo, and cytoplasm, of which maternal genetic effects were most important, followed by embryo and cytoplasmic genetic effects. The main effects of different genetic systems on IC trait were more important than environment interaction effects. The strong dominance effects on isoflavone from residual was made easily by environment conditions. Therefore,the improvement of the IC of soybean seeds would be more efficient when selection is based on maternal plants than that on the single seed. Maternal heritability (65.73%) was most important for IC, followed by embryo heritability (25.87%) and cytoplasmic heritability (8.39%). Based on predicated genetic effects, Yudou 29 and Zheng 90007 were better than other parents for increasing IC in the progeny and improving the quality of soybean. The significant effects of maternal and embryo dominance effects in variance show that the embryo heterosis and maternal heterosis are existent and uninfluenced by environment interaction effects.

Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

International Nuclear Information System (INIS)

Isobe, Ichiro; Maeno, Yoshitaka; Nagao, Masataka; Iwasa, Mineo; Koyama, Hiroyoshi; Seko-Nakamura, Yoshimi; Monma-Ohtaki, Jun

2003-01-01

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

CanVasc Recommendations for the Management of Antineutrophil Cytoplasm Antibody (ANCA-Associated Vasculitides – Executive Summary

Directory of Open Access Journals (Sweden)

Lucy McGeoch

2015-11-01

Full Text Available The Canadian Vasculitis research network (CanVasc is composed of physicians from different medical specialties, including rheumatology and nephrology and researchers with expertise in vasculitis. One of its aims was to develop recommendations for the diagnosis and management of antineutrophil cytoplasm antibody (ANCA-associated vasculitides in Canada. This executive summary features the 19 recommendations and 17 statements addressing general AAV diagnosis and management, developed by CanVasc group based on a synthesis of existing international guidelines, other published supporting evidence and expert consensus considering the Canadian healthcare context.

Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1976-01-01

The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

Structural changes in the cytoplasmic pore of the Kir1.1 channel during pHi-gating probed by FRET.

Science.gov (United States)

Lee, Jay-Ron; Shieh, Ru-Chi

2009-03-06

Kir1.1 channels are important in maintaining K+ homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K+ secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic pore changes in response to intracellular pH (pHi)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pHi pHi-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is completed closed at pHi 7.4 and open at pHi 10.0. FRET efficiency was increased from 25% to 40% when the pHi was decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel changes in response to pHi gating such that the N- and C-termini move apart from each other at pHi 7.4, when the channel is open.

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

Science.gov (United States)

Fiserova, Jindriska; Spink, Matthew; Richards, Shane A; Saunter, Christopher; Goldberg, Martin W

2014-01-01

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

DEFF Research Database (Denmark)

Weber, Roy E.

2007-01-01

Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the memb......Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain...... of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell...... membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. Methods: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3...

A sea urchin Na(+)K(+)2Cl(-) cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae.

Science.gov (United States)

Basse, Wiebke C; Gutowska, Magdalena A; Findeisen, Ulrike; Stumpp, Meike; Dupont, Sam; Jackson, Daniel J; Himmerkus, Nina; Melzner, Frank; Bleich, Markus

2015-09-01

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na(+)K(+)2Cl(-) cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50=6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4 μM) and furosemide (IC50=315.4 μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance. Copyright © 2015 Elsevier Inc. All rights reserved.

Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

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Travasso, Rui D M; Sampaio Dos Aidos, Fernando; Bayani, Anahita; Abranches, Pedro; Salvador, Armindo

2017-08-01

Hydrogen peroxide (H 2 O 2 ) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H 2 O 2 -reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion models of H 2 O 2 signaling. The results show that at H 2 O 2 supply rates commensurate with mitogenic signaling a H 2 O 2 concentration gradient with a length scale of a few tenths of μm is established. Even near the supply sites H 2 O 2 concentrations are far too low to oxidize typical targets in an early mitogenic signaling time frame. Furthermore, any inhibition of the peroxiredoxin or increase in H 2 O 2 supply able to drastically increase the local H 2 O 2 concentration would collapse the concentration gradient and/or cause an extensive oxidation of the peroxiredoxins I and II, inconsistent with experimental observations. In turn, the local concentrations of peroxiredoxin sulfenate and disulfide forms exceed those of H 2 O 2 by several orders of magnitude. Redox targets reacting with these forms at rate constants much lower than that for, say, thioredoxin could be oxidized within seconds. Moreover, the spatial distribution of the concentrations of these peroxiredoxin forms allows them to reach targets within 1 μm from the H 2 O 2 sites while maintaining signaling localized. The recruitment of peroxiredoxins to specific sites such as caveolae can dramatically increase the local concentrations of the sulfenic and disulfide forms, thus further helping these species to outcompete H 2 O 2 for the oxidation of redox targets. Altogether, these results suggest that H 2 O 2 signaling is mediated by localized redox relays whereby peroxiredoxins are oxidized to sulfenate and disulfide forms at H 2 O 2 supply

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase.

Science.gov (United States)

Šeflová, Jaroslava; Čechová, Petra; Štenclová, Tereza; Šebela, Marek; Kubala, Martin

2018-12-01

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na + /K + -ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

Analysing the impact of nucleo-cytoplasmic shuttling of β-catenin and its antagonists APC, Axin and GSK3 on Wnt/β-catenin signalling.

Science.gov (United States)

Schmitz, Yvonne; Rateitschak, Katja; Wolkenhauer, Olaf

2013-11-01

The canonical Wnt signalling pathway plays a critical role in development and disease. The key player of the pathway is β-catenin. Its activity is mainly regulated by the destruction complex consisting of APC, Axin and GSK3. In the nucleus, the complex formation of β-catenin and TCF initiates target gene expression. Our study provides a comprehensive analysis of the role of nucleo-cytoplasmic shuttling of APC, Axin, and GSK3 and the inactivation of β-catenin by the destruction complex in Wnt/β-catenin signalling. We address the following questions: Can nucleo-cytoplasmic shuttling of APC, Axin and GSK3 increase the [β-catenin/TCF] concentration? And, how is the [β-catenin/TCF] concentration influenced by phosphorylation and subsequent degradation of nuclear β-catenin? Based on experimental findings, we develop a compartmental model and conduct several simulation experiments. Our analysis reveals the following key findings: 1) nucleo-cytoplasmic shuttling of β-catenin and its antagonists can yield a spatial separation between the said proteins, which results in a breakdown of β-catenin degradation, followed by an accumulation of β-catenin and hence leads to an increase of the [β-catenin/TCF] concentration. Our results strongly suggest that Wnt signalling can benefit from nucleo-cytoplasmic shuttling of APC, Axin and GSK3, although they are in general β-catenin antagonising proteins. 2) The total robustness of the [β-catenin/TCF] output is closely linked to its absolute concentration levels. We demonstrate that the compartmental separation of β-catenin and the destruction complex does not only lead to a maximization, but additionally to an increased robustness of [β-catenin/TCF] signalling against perturbations in the cellular environment. 3) A nuclear accumulation of the destruction complex renders the pathway robust against fluctuations in Wnt signalling and against changes in the compartmental distribution of β-catenin. 4) Elucidating the impact of

Geologic Map of the Mylitta Fluctus Quadrangle (V-61), Venus

Science.gov (United States)

Ivanov, Mikhail A.; Head, James W.

2006-01-01

INTRODUCTION The Magellan Mission The Magellan spacecraft orbited Venus from August 10, 1990, until it plunged into the Venusian atmosphere on October 12, 1994. Magellan Mission objectives included: (1) improving knowledge of the geological processes, surface properties, and geologic history of Venus by analysis of surface radar characteristics, topography, and morphology, and (2) improving the knowledge of the geophysics of Venus by analysis of Venusian gravity. The Magellan spacecraft carried a 12.6-cm radar system to map the surface of Venus. The transmitter and receiver systems were used to collect three data sets: (1) synthetic aperture radar (SAR) images of the surface, (2) passive microwave thermal emission observations, and (3) measurements of the backscattered power at small angles of incidence, which were processed to yield altimetric data. Radar imaging, altimetric, and radiometric mapping of the Venusian surface was done in mission cycles 1, 2, and 3 from September 1990 until September 1992. Ninety-eight percent of the surface was mapped with radar resolution on the order of 120 meters. The SAR observations were projected to a 75-m nominal horizontal resolution, and these full-resolution data compose the image base used in geologic mapping. The primary polarization mode was horizontal-transmit, horizontal-receive (HH), but additional data for selected areas were collected for the vertical polarization sense. Incidence angles varied between about 20? and 45?. High resolution Doppler tracking of the spacecraft took place from September 1992 through October 1994 (mission cycles 4, 5, 6). Approximately 950 orbits of high-resolution gravity observations were obtained between September 1992 and May 1993 while Magellan was in an elliptical orbit with a periapsis near 175 km and an apoapsis near 8,000 km. An additional 1,500 orbits were obtained following orbit-circularization in mid-1993. These data exist as a 75? by 75? harmonic field.

Intraspecific variation in mitochondrial genome sequence, structure, and gene content in Silene vulgaris, an angiosperm with pervasive cytoplasmic male sterility

Czech Academy of Sciences Publication Activity Database

Sloan, D.B.; Müller, Karel; McCauley, D.; Taylor, D.R.; Štorchová, Helena

2012-01-01

Roč. 196, č. 4 (2012), s. 1228-1239 ISSN 0028-646X R&D Projects: GA ČR GA521/09/0261; GA MŠk(CZ) LC06004; GA MŠk ME09035 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytoplasmic male sterility (CMS) * gynodioecy * intracellular gene transfer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.736, year: 2012

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta

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Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena

2014-01-01

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS). PMID:25046115

No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects.

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Janne M Toivonen

2007-06-01

Full Text Available To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans.

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

Science.gov (United States)

Heude, M; Chanet, R

1975-04-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Science.gov (United States)

Balsamo, Janne; Arregui, Carlos; Leung, TinChung; Lilien, Jack

1998-01-01

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion. PMID:9786960

Degradation of YRA1 Pre-mRNA in the cytoplasm requires translational repression, multiple modular intronic elements, Edc3p, and Mex67p.

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Shuyun Dong

2010-04-01

Full Text Available Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron. Two of these elements target the pre-mRNA as an Edc3p substrate and the other three mediate transcript-specific translational repression. Translational repression of YRA1 pre-mRNA also requires the heterodimeric Mex67p/Mtr2p general mRNA export receptor, but not Edc3p, and serves to enhance Edc3p substrate specificity by inhibiting the susceptibility of this pre-mRNA to NMD. Collectively, our data indicate that YRA1 pre-mRNA degradation is a highly regulated process that proceeds through translational repression, substrate recognition by Edc3p, recruitment of the Dcp1p/Dcp2p decapping enzyme, and activation of decapping.

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS

DEFF Research Database (Denmark)

Scharff, O; Foder, B; Thastrup, Ole

1988-01-01

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full...... of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects...

Whole Mitochondrial Genome Sequencing and Re-Examination of a Cytoplasmic Male Sterility-Associated Gene in Boro-Taichung-Type Cytoplasmic Male Sterile Rice.

Science.gov (United States)

Kazama, Tomohiko; Toriyama, Kinya

2016-01-01

Nuclear genome substitutions between subspecies can lead to cytoplasmic male sterility (CMS) through incompatibility between nuclear and mitochondrial genomes. Boro-Taichung (BT)-type CMS rice was obtained by substituting the nuclear genome of Oryza sativa subsp. indica cultivar Chinsurah Boro II with that of Oryza sativa subsp. japonica cultivar Taichung 65. In BT-type CMS rice, the mitochondrial gene orf79 is associated with male sterility. A complete sequence of the Boro-type mitochondrial genome responsible for BT-type CMS has not been determined to date. Here, we used pyrosequencing to construct the Boro-type mitochondrial genome. The contiguous sequences were assembled into five circular DNA molecules, four of which could be connected into a single circle. The two resulting subgenomic circles were unable to form a reliable master circle, as recombination between them was scarcely detected. We also found an unequal abundance of DNA molecules for the two loci of atp6. These results indicate the presence of multi-partite DNA molecules in the Boro-type mitochondrial genome. Expression patterns were investigated for Boro-type mitochondria-specific orfs, which were not found in the mitochondria from the standard japonica cultivar Nipponbare. Restorer of fertility 1 (RF1)-dependent RNA processing has been observed in orf79-containing RNA but was not detected in other Boro-type mitochondria-specific orfs, supporting the conclusion that orf79 is a unique CMS-associated gene in Boro-type mitochondria.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

International Nuclear Information System (INIS)

Quaresma, Alexandre J.C.; Bressan, G.C.; Gava, L.M.; Lanza, D.C.F.; Ramos, C.H.I; Kobarg, Joerg

2009-01-01

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 - GWbs marker protein and TIA-1 stress granule component

Herpes Simplex Virus 1 Us3 Deletion Mutant is Infective Despite Impaired Capsid Translocation to the Cytoplasm

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Peter Wild

2015-01-01

Full Text Available Herpes simplex virus 1 (HSV-1 capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i The number of R7041(∆US3 capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii The mean number of R7041(∆US3 virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii 98% of R7041(∆US3 virions were in the perinuclear space; (iv The number of R7041(∆US3 capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3 yields were 2.37 × 108 and HSV-1 yields 1.57 × 108 PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3 virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Observation of a cytoplasmic inclusion in peripheral blood of a-bomb survivors

International Nuclear Information System (INIS)

Neriishi, Shotaro; Ohgushi, Yasutaka; Sadamori, Naoki.

1978-01-01

Whether cytoplasmic inclusion (CI) is useful as an index for the effects of atomic bomb radiation was discussed. Subjects of this study were 16 healthy people with chromosomal aberration due to atomic bomb radiation (8 males and 8 females) (Group 1) and 31 people with a history of malignant tumors (10 males and 21 females) (Group 2) who were living in Nagasaki City. For the purpose of observing chromosome and CI, blood was collected together in the period from 1970 to 1972. In Group 1, a total number of cells obseved was 14,513 and no Cl was found in them. In Group 2, a total number of cells observed was 31,289, and CI was found in 2 of them. CI which is a reaction of a living body in an acute stage following accidental irradiation seemed to have already disappeared. Therefore, usefulness of CI as an indicator for the effect of atomic bomb radiation was denied. (Ueda, J.)

Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8.

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Yen-Chin Liu

2014-06-01

Full Text Available The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D(pol also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D(pol enters the nucleus through the nuclear localization signal (NLS and targets the pre-mRNA processing factor 8 (Prp8 to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D(pol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D(pol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.

Correlation of cytoplasmic beta-catenin and cyclin D1 overexpression during thyroid carcinogenesis around Semipalatinsk nuclear test site.

Science.gov (United States)

Meirmanov, Serik; Nakashima, Masahiro; Kondo, Hisayoshi; Matsufuji, Reiko; Takamura, Noboru; Ishigaki, Katsu; Ito, Masahiro; Prouglo, Yuri; Yamashita, Shunichi; Sekine, Ichiro

2003-06-01

The Semipalatinsk nuclear test site (SNTS), the Republic of Kazakhstan, has been contaminated by radioactive fallout. The alteration of oncogenic molecules in thyroid cancer around the SNTS was considered worthy of analysis because it presented the potential to elucidate the relationship between radiation exposure and thyroid cancer. This study aimed to analyze both beta-catenin and cyclin D1 expressions in thyroid carcinomas around the SNTS. We examined nine cases of chronic thyroiditis, eight cases of follicular adenomas, and 23 cases of papillary carcinomas. Immunohistochemically, all carcinomas displayed a strong cytosolic beta-catenin expression, while both chronic thyroiditis and follicular adenomas showed a significantly lower cytoplasmic beta-catenin (22.2% and 37.5%, respectively). No cyclin D1 immunoreactivity was evident in chronic thyroiditis. In contrast, 62.5% of follicular adenomas and 87.0% of papillary carcinoma showed cyclin D1 overexpression. Additionally, a strong correlation between cytoplasmic beta-catenin and cyclin D1 expression was suggested in thyroid tumors. This study revealed a higher prevalence of both aberrant beta-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the SNTS than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the SNTS may be important to gain an insight into radiation-induced thyroid tumorigenesis.

Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

Science.gov (United States)

Layhadi, Janice A; Fountain, Samuel J

2017-06-03

Mechanisms controlling endoplasmic reticulum (ER) Ca 2+ homeostasis are important regulators of resting cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ) and receptor-mediated Ca 2+ signalling. Here we investigate channels responsible for ER Ca 2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca 2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca 2+ leak. Under these conditions ionomycin elevates [Ca 2+ ] cyto revealing a Ca 2+ leak response which is abolished by thapsigargin. IP 3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca 2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca 2+ ] cyto . In primary macrophage, translocon inhibition blocks Ca 2+ leak but does not influence resting [Ca 2+ ] cyto . We identify a role for translocon-mediated ER Ca 2+ leak in receptor-mediated Ca 2+ signalling in both model and primary human macrophage, whereby the Ca 2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca 2+ leak via the translocon in controlling resting cytoplasmic Ca 2+ in model macrophage and receptor-mediated Ca 2+ signalling in model macrophage and primary macrophage. Copyright © 2017 Elsevier Inc. All rights reserved.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

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Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

1993-03-01

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes

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Caroline Vindry

2017-08-01

Full Text Available Pat1 RNA-binding proteins, enriched in processing bodies (P bodies, are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA. Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase.

Science.gov (United States)

Bamford, Vicki A; Armour, Maria; Mitchell, Sue A; Cartron, Michaël; Andrews, Simon C; Watson, Kimberly A

2008-09-01

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Coulomb interactions between cytoplasmic electric fields and phosphorylated messenger proteins optimize information flow in cells.

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Robert A Gatenby

2010-08-01

Full Text Available Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM. While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl- and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length.Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions.This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger

[Isolation and identification of specific sequences correlated to cytoplasmic male sterility and fertile maintenance in cauliflower (Brassica oleracea var. botrytis)].

Science.gov (United States)

Wang, Chun Guo; Chen, Xiao Qiang; Li, Hui; Zhao, Qian Cheng; Sun, De Ling; Song, Wen Qin

2008-02-01

Analysis of ISSR (Inter-Simple Sequence Repeat) and DDRT-PCR (Differential Display Reverse Transcriptase Polymerase Chain Reaction) was performed between cytoplasmic male sterility cauliflower ogura-A and its corresponding maintainer line ogura-B. Totally, 306 detectable bands were obtained by ISSR using thirty oligonucleotide primers. Commonly, six to twelve bands were produced per primer. Among all these primers only the amplification of primer ISSR3 was polymorphic, an 1100 bp specific band was only detected in maintainer line, named ISSR3(1100). Analysis of this sequence indicated that ISSR3(1100) was high homologous with the corresponding sequences of mitochondrial genome in Brassica napus and Arabidopsis thaliana,which suggested that ISSR3(1100) may derive from mitochondrial genome in cauliflower. To carry out DDRT-PCR analysis, three anchor primers and fifteen random primers were selected to combine. Totally, 1122 bands from 1 000 bp to 50 bp were detected. However, only four bands, named ogura-A 205, ogura-A383, ogura-B307 and ogura-B352, were confirmed to be different display in both lines. This result was further identified by reverse Northern dot blotting analysis. Among these four bands, ogura-A205 and ogura-A383 only express in cytoplasmic male sterility line, while ogura-B307 and ogura-B352 were only detected in maintainer line. Analysis of these sequences indicated that it was the first time that these four sequences were reported in cauliflower. Interestingly, ogura-A205 and ogura-B307 did not exhibit any similarities to other reported sequences in other species, more investigations were required to obtain further information. ogura-A383 and ogura-B352 were also two new sequences, they showed high similarities to corresponding chloroplast sequences of Arabidopsis thaliana and Brassica rapa subsp. pekinensis. So we speculated that these two sequences may derive from chloroplast genome. All these results obtained in this study offer new and

Retromer associates with the cytoplasmic amino-terminus of polycystin-2.

Science.gov (United States)

Tilley, Frances C; Gallon, Matthew; Luo, Chong; Danson, Chris M; Zhou, Jing; Cullen, Peter J

2018-05-03

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic human disease, with around 12.5 million people affected worldwide. ADPKD results from mutations in either PKD1 or PKD2 , which encode the atypical G-protein coupled receptor polycystin-1 (PC1) and the transient receptor potential channel polycystin-2 (PC2) respectively. Although altered intracellular trafficking of PC1 and PC2 appear as an underlying feature of ADPKD, the mechanisms which govern vesicular transport of the polycystins through the biosynthetic and endosomal membrane networks remain to be fully elucidated. Here, we describe an interaction between PC2 and retromer, a master controller for the sorting of integral membrane proteins through the endo-lysosomal network. We show that association of PC2 with retromer occurs via a region in the PC2 cytoplasmic amino-terminal domain, independently of the retromer-binding Wiskott-Aldrich syndrome and scar homologue (WASH) complex. Based on observations that retromer preferentially interacts with a trafficking population of PC2, and that ciliary levels of PC1 are reduced upon mutation of key residues required for retromer-association in PC2, our data is consistent with the identification of PC2 as a retromer cargo protein. © 2018. Published by The Company of Biologists Ltd.

The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix

DEFF Research Database (Denmark)

Wueseke, Oliver; Bunkenborg, Jakob; Hein, Marco Y

2014-01-01

mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead...

Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

Science.gov (United States)

Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

2011-07-01

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

Exclusion of NFAT5 from mitotic chromatin resets its nucleo-cytoplasmic distribution in interphase.

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Anaïs Estrada-Gelonch

Full Text Available BACKGROUND: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5. METHODOLOGY/PRINCIPAL FINDINGS: Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD. NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin. CONCLUSIONS/SIGNIFICANCE: Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export

Lessons from Animal Models of Cytoplasmic Intermediate Filament Proteins.

Science.gov (United States)

Bouameur, Jamal-Eddine; Magin, Thomas M

Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies. Epidermal keratins, together with desmin, are essential to protect corresponding tissues against mechanical force but also participate in stabilizing cell adhesion and in inflammatory signalling. Surprisingly, other IF proteins contribute to tissue integrity to a much lesser extent than anticipated, pointing towards their role in stress situations. In support, the overexpression of small chaperones or the interference with inflammatory signalling in several settings has been shown to rescue severe tissue pathologies that resulted from the expression of mutant IF proteins. It stills remains an open issue whether the wide range of IF disorders share similar pathomechanisms. Moreover, we lack an understanding how IF proteins participate in signalling processes. Now, with a large number of mouse models in hand, the next challenge will be to develop organotypic cell culture models to dissect pathomechanisms at the molecular level, to employ Crispr/Cas-mediated genome engineering to optimize models and, finally, to combine available animal models with medicinal chemistry for the development of molecular therapies.

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

International Nuclear Information System (INIS)

Patheja, Pooja; Sahu, Khageswar

2017-01-01

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

Energy Technology Data Exchange (ETDEWEB)

Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

2017-06-15

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.